U.S. patent number 5,569,601 [Application Number 07/928,611] was granted by the patent office on 1996-10-29 for human dopamine receptor and its uses.
This patent grant is currently assigned to State of Oregon, Acting by and Through the Oregon State Board of Higher. Invention is credited to Olivier Civelli.
United States Patent |
5,569,601 |
Civelli |
October 29, 1996 |
Human dopamine receptor and its uses
Abstract
The present invention is directed toward the isolation,
characterization and pharmacological use of the human D4 dopamine
receptor. The nucleotide sequence of the gene corresponding to this
receptor and alleleic variant thereof are provided by the
invention. The invention also includes recombinant eukaryotic
expression constructs capable of expressing the human D4 dopamine
receptor in cultures of transformed eukaryotic cells. The invention
provides cultures of transformed eukaryotic cells which synthesize
the human D4 dopamine receptor, and methods for characterizing
novel psychotropic compounds using such cultures.
Inventors: |
Civelli; Olivier (Portland,
OR) |
Assignee: |
State of Oregon, Acting by and
Through the Oregon State Board of Higher (Portland,
OR)
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Family
ID: |
25456529 |
Appl.
No.: |
07/928,611 |
Filed: |
August 10, 1992 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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626618 |
Dec 7, 1990 |
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Current U.S.
Class: |
435/365;
435/320.1; 435/69.1; 435/71.1; 536/23.5; 930/50 |
Current CPC
Class: |
C07K
14/70571 (20130101); C12Q 1/683 (20130101); G01N
33/9406 (20130101); G01N 33/9413 (20130101); G01N
2500/00 (20130101); G01N 2500/10 (20130101); G01N
2800/52 (20130101) |
Current International
Class: |
C07K
14/435 (20060101); C07K 14/705 (20060101); C12Q
1/68 (20060101); G01N 33/94 (20060101); C12N
005/00 (); C12N 015/00 (); C12P 021/06 (); C07H
017/00 () |
Field of
Search: |
;536/23.5,24.31
;435/240.2,320.1,69.1,71.1,172.1,172.3 ;935/22,23,52,55
;930/50 |
Foreign Patent Documents
Other References
Van Tol et al., Nature, vol. 350, 18 Apr. 1991, pp. 610-614. .
Van Tol et al., Nature, vol. 358, 9 Jul. 1992, pp. 149-152. .
Botstein et al., 1980, Am. J. Hum. Genet. 32:314-331. .
Van Tol et al., 1991, Nauture 350:610-614. .
Cooper et al., The Biochemical Basis of Neuropharmacology, 3d ed.
1978 (Oxford University Press: N.Y.), pp. 161-195. .
Kababian and Calna, Nature 277:93-96 (1979). .
Sanogles et al., Biochemistry 25:749-753 (1986). .
Sangoles et al., J. Biol. Chem. 263:18996-19002 (1988). .
Gingrich et al., J. Biochemistry 27:3907-3912 (1988). .
Amlaiky et al., Mol. Pharmacol. 31:129-134 (1987). .
Ninik et al., Biochemistry 27:7594-7599 (1988). .
Amlaiky and Caron, J. Biol. Chem. 260:1983-1986 (1985). .
Amlaiky and Caron, J. Neurohem. 47:196-204 (1986). .
Jarvia et al., Mol. Pharmacol. 34:91-97 (1988). .
Sokoloff et al., Nature 347:146-151 (1990). .
Seaman et al., Neuropsychopharm. 1:5-15 (1987). .
Seaman, Synapse 1:133-152 (1987). .
Bunaew et al., Nature 336:783-787 (1988). .
Grandy et al., Proc. Natl. Acad. Sci. U.S.A. 86:9762-9766 (1989).
.
Dal Toso et al., Embo. J. 8:4025-4034 (1989). .
Zhou et al., Nature 348:76-80 (1990). .
Sunahara et al., Nature 348:80-83 (1990). .
Kane et al., Arch. Gan. Psychiat. 45:789-796 (1988). .
Casey, Psychopharmacology 99:547-553 (1989). .
Ackenheil et al., Arzneim-Forsch 26:1156-1158 (1976). .
Bandoz Canada, Inc., Clozaril: Summary of preclinical and clinical
data (1990). .
Dohlman et al., Biochemistry 26:2657-2664 (1987). .
Botstein et al., 1980, Am. J. Hum. Genet. 32:314-331. .
Van Tol et al., 1991, Nature 350:610-614. .
Van Tol et al., 1992, Nature 368:149-152..
|
Primary Examiner: Parr; Margaret
Assistant Examiner: Sisson; Bradley L.
Attorney, Agent or Firm: Banner & Allegretti, Ltd.
Government Interests
This invention was made with government support under NIMH grant
MH-45614 awarded by the National Institutes of Health, Unites
States of America, and grant PG 11121 awarded by the Medical
Research Council of Canada. The governments have certain rights in
the invention.
Parent Case Text
This application is a continuation-in-part of U.S. patent
application Ser. No. 07/626,618, filed on Dec. 7, 1990, which is
hereby incorporated by reference.
Claims
What we claim is:
1. An isolated nucleic acid having a nucleotide residue sequence
encoding an amino acid residue sequence selected from the group
consisting of the human D.sub.4 dopamine receptor allees D4.4 (SEQ
ID No.: 20) and D4.7 (SEQ ID No.: 22).
2. A recombinant expression construct comprising a nucleic acid
having a nucleotide sequence encoding a human D.sub.4 dopamine
receptor according to claim 1.
3. A eukaryotic cell culture transformed with the recombinant
expression construct of claim 2, wherein the transformed eukaryotic
cell culture is capable of expressing the human dopamine receptor
D4.
4. An isolated and purified nucleic acid having a nucleotide
residue sequence encoding an amino acid residue sequence, wherein
the nucleotide residue sequence consists of a 5' sequence encoding
the amino acid residue sequence of the human D.sub.4 dopamine from
amino acid 1 to amino acid 248 of SEQ ID NO.: 20, a repeated
sequence and a 3' sequence encoding the amino acid residue sequence
of the human D.sub.4 dopamine receptor allele D4.4 from amino acid
312 to amino acid 419 of SEQ ID NO.: 20, and wherein the nucleotide
sequence of the repeated sequence comprises from 3 to 8 copies of a
nucleotide residue sequence encoding the amino acid sequence:
(Pro/Ala).Ala/Pro.(Arg/Ser/Gly).Leu.Pro.(Gln/Arg/Pro).(Asp/Gly).Pro.Cys.Gl
y.(Pro/Ser).(Asp/Asn).Cys.Ala/Pro of SEQ ID NO. 20.
5. A recombinant expression construct comprising the nucleic acid
of claim 4.
6. The recombinant expression construct of claim 5 comprising from
3 to 8 copies of the repeated DNA sequence.
7. A eukaryotic cell culture transformed with the recombinant
expression construct of claim 5, wherein the transformed eukaryotic
cell culture is capable of expressing the human dopamine receptor
D4.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to dopamine receptors from mammalian species
and the genes corresponding to such receptors. In particular, it
relates to the human dopamine receptor D4. Specifically, the
invention relates to the isolation, cloning and sequencing of the
human D4 receptor gene. The invention also relates to the
construction of eukaryotic expression vectors capable of expression
of the human D4 dopamine receptor in cultures of transformed
eukaryotic cells and the synthesis of the human D4 dopamine
receptor in such cultures. The invention relates to the use of such
cultures of transformed eukaryotic cells producing the human D4
dopamine receptor for the characterization of antipsychotic
drugs.
2. Information Disclosure Statement
Dopamine is a neurotransmitter that participates in a variety of
different functions mediated by the nervous system, including
vision, movement, and behavior (see generally Cooper et al., 1978,
The Biochemical Basis of Neuropharmacology, 3d ed., Oxford
University Press, New York, pp. 161-195). The diverse physiological
actions of dopamine are in turn mediated by its interaction with
two of the basic types of G protein-coupled receptors, D1 and D2,
which respectively stimulate and inhibit the enzyme adenylyl
cyclase (Kebabian & Calne, 1979, Nature 277: 93-96).
Alterations in the number or activity of these receptors may be a
contributory factor in disease states such as Parkinson's disease
(a movement disorder) and schizophrenia (a behavioral
disorder).
A great deal of information has accumulated on the biochemistry of
the D1 and D2 dopamine receptors, and methods have been developed
to solubilize and purify these receptor proteins (see Senogles et
al., 1986, Biochemistry 25: 749-753; Sengoles et al., 1988, J.
Biol. Chem. 263: 18996-19002; Gingrich et al., 1988, Biochemistry
27: 3907-3912). The D1 dopamine receptor in several tissues appears
to be a glycosylated membrane protein of about 72 kD (Amlaiky et
al., 1987, Mol. Pharmacol. 31: 129-134; Ninik et al., 1988,
Biochemistry 27: 7594-7599). The D2 receptor has been suggested to
have a higher molecular weight of about 90-150 kD (Amlaiky &
Caron, 1985, J. Biol. Chem. 260: 1983-1986; Amlaiky & Caron,
1986, J. Neurochem. 47: 196-204; Jarvie et al., 1988, Mol.
Pharmacol. 34: 91-97). Much less is known about a recently
discovered additional dopamine receptor, termed D3 (Sokoloff et
al., 1990, Nature 347: 146-151).
Dopamine receptors are primary targets in the clinical treatment of
psychomotor disorders such as Parkinson's disease and affective
disorders such as schizophrenia (Seeman et al., 1987,
Neuropsychopharm. 1: 5-15; Seeman, 1987, Synapse 1: 152-333). The
three different dopamine receptors (D1, D2, D3) have been cloned as
a result of nucleotide sequence homology which exists between these
receptor genes (Bunzow et al., 1988, Nature 336: 783-787; Grandy et
al., 1989, Proc. Natl. Acad. Sci. USA 86: 9762-9766; Dal Toso et
al., 1989, EMBO J. 8: 4025-4034; Zhou et al., 1990, Nature 346:
76-80; Sunahara et al., 1990, Nature 346: 80-83; Sokoloff et al.,
1990, Nature 347: 146-151).
The antipsychotic clozapine is useful for socially withdrawn and
treatment-resistant schizophrenics (see Kane et al., 1990, Nature
347: 146-151), but unlike other antipsychotic drugs, clozapine does
not cause tardive dyskinesia (see Casey, 1989, Psychopharmacology
99: 547-553). Clozapine, however, has dissociation constants for D2
and D3 which are 3 to 30-fold higher than the therapeutic free
concentration of clozapine in plasma water (Ackenheil et al., 1976,
Arzneim-Forsch 26: 1156-1158; Sandoz Canada, Inc., 1990, Clozaril:
Summary of preclinical and clinical data). This suggests the
existence of dopamine receptors more sensitive to the antipsychotic
clozapine than those known in the prior art heretofore.
We have cloned and sequenced such a human dopamine receptor which
we term D4. The dopamine D4 receptor gene has high homology to the
human dopamine D2 and D3 receptor genes. The pharmacological
profile of this receptor resembles that of the D2 and D3 receptors
but it has an affinity for clozapine which is tenfold higher. The
present inventors envision that the D4 dopamine receptor disclosed
as this invention may prove useful in discovering new types of
drugs for schizophrenia that like clozapine do not induce tardive
dyskinesia and other motor side effects.
We have also discovered that the D4 gene is polymorphic in the
human population, having at least 7 different alleles that can be
detected by restriction fragment length polymorphism analysis (see,
Botstein et al., 1980, Am. J. Hum. Genet. 32: 314-331). This is the
first receptor in the catecholamine receptor family which displays
polymorphic variations in the human population. The observed
polymorphism in dopamine D4 receptor genes may underlie individual
differences in susceptibility to neuropsychiatric disorders such as
schizophrenia and manic depression, as well as responsiveness to
antipsychotic medication.
DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the structure of a genomic clone comprising the
human D4 dopamine receptor gene.
FIGS. 2A through 2D illustrates the nucleotide sequence of genomic
and cDNA clones of the human D4 topamine receptor gene.
FIGS. 3 and 3A through 3F provides an amino acid sequence alignment
of mammalian dopamine receptors
FIGS. 4A and 4B shows the binding of [.sup.3 H]spiperone to
membranes of COS-7 cell transfected with a recombinant expression
construct that expresses the human D4 receptor protein.
FIG. 5 demonstrates the pharmacological specificity of [.sup.3
H]spiperone binding to COS-7 cells transfected with a human D4
receptor expression construct.
FIGS. 6A through 6C illustrates the structure of a genomic clone
comprising the human D4 dopamine receptor gene and the nucleic acid
and corresponding amino acid sequences of 2, 4 and 7 copies of a
novel 48 bp tandem repeat.
FIG. 7 illustrates restriction fragment length polymorphic variants
of the human D4 receptor gene in 9 individuals.
FIG. 8 demonstrates the transcriptional integrity of each of three
cloned variant human D4 receptor gene expression constructs
expressed in transfected COS-7 cells.
FIGS. 9A through 9C illustrate Scatchard analysis of each of three
cloned variant human D4 receptor gene expression constructs
expressed in transfected COS-7 cell.
FIGS. 9D through 9F illustrate [.sup.3 H]-spiperone competition
binding experiments of each of three cloned variant human D4
receptor gene expression constructs expressed in transfected COS-7
cell.
SUMMARY OF THE INVENTION
The present invention is directed toward the isolation,
characterization and pharmacological use of the human D4 dopamine
receptor, the gene corresponding to this receptor, a recombinant
eukaryotic expression construct capable of expressing the human D4
dopamine receptor in cultures of transformed eukaryotic cells and
such cultures of transformed eukaryotic cells that synthesize the
human D4 dopamine receptor.
It is an object of the invention to provide a nucleotide sequence
encoding a mammalian dopamine receptor. Further, it is an object of
the invention to provide a nucleotide sequence that encodes a
mammalian dopamine receptor with novel and distinct pharmacological
properties. It is specifically an object of the invention to
provide a nucleotide sequence encoding a mammalian dopamine
receptor having the particular drug dissociation properties of the
human dopamine receptor D4. In particular, the mammalian dopamine
receptor encoded by the nucleotide sequence of the present
invention has a high affinity for the drug clozapine. The human D4
dopamine receptor embodied in the present invention shows a
dissociation constant (termed K.sub.i) of 1-40 nanomolar (nM),
preferably 1-20 nM, most preferably 11 nM clozapine, as detected by
the [.sup.3 H]spiperone binding assay disclosed herein. The human
D4 dopamine receptor embodied in the present invention displays the
following pharmacological profile of inhibition of [.sup.3
H]spiperone binding in the [.sup.3 H]spiperone binding assay:
spiperone>eticlopride>clozapine>(+)-butaclamol>racloopride>SCH23390.
In a preferred embodiment of the invention, the nucleotide sequence
encoding a dopamine receptor encodes the human dopamine receptor
D4.
The present invention provides a nucleotide sequence encoding a
mammalian dopamine receptor that is the human D4 receptor. In a
preferred embodiment, this nucleotide sequence comprises a cDNA
sequence isolated from RNA derived from the human neuroblastoma
cell line SK-N-MC [SEQ ID No: 17], comprising the sequences of the
D4.2 allele of the human D4 dopamine receptor gene. In another
preferred embodiment, this nucleotide sequence comprises a cDNA
sequence isolated from RNA derived from human pituitary gland
tissue [SEQ ID No: 19]. In yet another preferred embodiment, this
nucleotide sequence comprises a cDNA sequence isolated from RNA
derived from human substantia nigra tissue [SEQ ID No.: 21]. Both
of these embodiments comprise the sequences of the D4.4 allele of
the human D4 dopamine receptor gene.
The invention also includes a nucleotide sequence derived from
human genomic DNA [SEQ ID Nos.: 1,3,4,5,7,12,14 & 15]
comprising the sequences of the D4.7 allele of the human D4
dopamine receptor gene, and a nucleotide sequence derived from
human genomic DNA [SEQ ID Nos.: 1,3,4,5,7,10,14 & 15]
comprising the sequences of the D4.4 allele of the human D4
dopamine receptor gene. In this embodiment of the invention, the
nucleotide sequence includes 5 kilobases (kb) of human genomic DNA
encoding the dopamine receptor D4. This embodiment includes the
sequences present in the cDNA embodiments as well as nucleotide
sequences of 5' untranslated sequence, three intervening sequences
that interrupt the coding sequence of the human D4 dopamine
receptor gene, and 3' untranslated sequences. Also provided is a
cDNA sequence derived from the genomic DNA sequence of the D4.4.
allele [SEQ ID No: 19] and the D4.7 allele [SEQ ID No: 21] of the
human D4 dopamine receptor gene.
The invention includes a nucleotide sequence of a human D4 receptor
molecule, and includes allelic variations of this nucleotide
sequence and the corresponding D4 receptor molecule, either
naturally occurring or the product of in vitro chemical or genetic
modification, having essentially the same nucleotide sequence as
the nucleotide sequence of the human D4 receptor disclosed herein,
wherein the resulting human D4 receptor molecule has substantially
the same drug dissociation properties of the human D4 receptor
molecule corresponding to the nucleotide sequence described herein.
Specific preferred embodiments include alleles D4.2, D4.4 and D4.7
of the human D4 dopamine receptor gene, as defined herein.
The invention provides sequences of the naturally-occurring alleles
of the human D4 dopamine receptor gene. Such alleles are defined as
comprising from about 2 to about 8 repeats of a nucleotide sequence
that is substantially homologous to the sequence [SEQ ID Nos:
8,10,12,17,19,21]:
Allelic variations of this nucleotide sequence and the
corresponding D4 receptor molecule, either naturally occurring or
the product of in vitro chemical or genetic modification, having
essentially the same nucleotide sequence as the nucleotide sequence
of the human D4 receptor disclosed herein, wherein the resulting
human D4 receptor molecule has substantially the same drug
dissociation properties of the human D4 receptor molecule
corresponding to the nucleotide sequence described herein are
additional preferred embodiments of the invention. Specific
preferred embodiments include the allele D4.2, comprising 2 copies
of the repeat tandemly repeated [SEQ ID Nos: 8 & 17]; the
allele D4.4, comprising 4 copies of the repeat tandemly repeated
[SEQ ID Nos: 10 & 19]; and the allele D4.7, comprising 7 copies
of the repeat tandemly repeated [SEQ ID Nos: 12 & 21].
The invention also includes a predicted amino acid sequence for the
human D4 dopamine receptor deduced from the nucleotide sequence
comprising the complete coding sequence of the D4 dopamine receptor
gene [SEQ ID Nos: 18, 20 & 22]. Specific preferred embodiments
comprise the amino acid sequence of the naturally-occurring alleles
of the human D4 dopamine receptor gene. Such alleles are defined as
comprising from about 2 to about 8 repeats of an amino acid
sequence that is substantially homologous to the sequence [SEQ ID
Nos: 9,11,13,18,20,22]:
Allelic variations of this amino acid and the corresponding D4
receptor molecule, either naturally occurring or the product of in
vitro chemical or genetic modification, having essentially the same
amino acid sequence as the human D4 receptor disclosed herein,
wherein the human D4 receptor molecule has substantially the same
drug dissociation properties of the human D4 receptor molecule
corresponding to the amino acid sequence described herein are
additional preferred embodiments of the invention. Specific
preferred embodiments include the allele D4.2, comprising 2 copies
of the repeat tandemly repeated [SEQ ID Nos: 9 & 18]; the
allele D4.4, comprising 4 copies of the repeat tandemly repeated
[SEQ ID Nos: 11 & 20]; and the allele D4.7, comprising 7 copies
of the repeat tandemly repeated [SEQ ID Nos: 13 & 22].
This invention provides both nucleotide and amino acid probes
derived from these sequences. The invention includes probes
isolated from either cDNA or genomic DNA clones, as well as probes
made synthetically with the sequence information derived therefrom.
The invention specifically includes but is not limited to
oligonucleotide, nick-translated, random primed, or in vitro
amplified probes made using cDNA or genomic clones embodying the
invention, and oligonucleotide and other synthetic probes
synthesized chemically using the nucleotide sequence information of
cDNA or genomic clone embodiments of the invention. The sequence
information provided by the present invention is also intended to
provide the basis for in vitro amplification methods for detecting
D4 dopamine receptor alleles comprising the genotype of somatic and
germ cells, zygotes, embryoes, and tissues in humans and other
mammals for diagnostic, therapeutic and other purposes.
It is a further object of this invention to provide sequences of
the human D4 dopamine receptor for use as probes to determine the
pattern, amount and extent of expression of this receptor in
various tissues of mammals, including humans. It is also an object
of the present invention to provide probes derived from the
sequences of the human D4 dopamine receptor to be used for the
detection and diagnosis of genetic diseases. It is an object of
this invention to provide probes derived from the sequences of the
human D4 dopamine receptor to be used for the detection of novel
related receptor genes.
The present invention also includes synthetic peptides made using
the nucleotide sequence information comprising the cDNA or genomic
clone embodiments of the invention. The invention includes either
naturally occurring or synthetic peptides which may be used as
antigens for the production of D4 dopamine receptor-specific
antibodies, or used for competitors of the D4 receptor molecule for
drug binding, or to be used for the production of inhibitors (or
blockers) of the binding of dopamine or dopamine analogs of the D4
dopamine receptor molecule. As used herein, the term "inhibitor of
dopamine binding" is intended to encompass biochemical agonists
and/or antagonists of dopamine binding to the D4 dopamine
receptor.
In addition, this invention includes recombinant DNA constructs
comprising the human D4 dopamine receptor and sequences that
mediate the replication and selected growth of microorganisms that
carry this construct.
The present invention provides recombinant expression constructs
comprising the nucleotide sequence of the human D4 dopamine
receptor and sequences sufficient to direct the synthesis of the
human D4 dopamine receptor protein in cultures of transformed
eukaryotic cells. In preferred embodiments, the recombinant
expression construct is comprised of plasmid sequences derived from
the plasmid pCD-PS and D4 dopamine receptor sequences corresponding
to cDNA sequences for alleles D4.2, D4.4 and D4.7, as defined
herein, as well as a hybrid human D4 dopamine gene, comprised of
the entirety of the genomic sequences from a particular D4 dopamine
genomic clone described herein, up to a PstI site located in exon
III, followed by the remainder of the coding and 3' untranslated
sequences found in a particular human cDNA sequence derived from a
human neuroblastoma cell line. Recombinant expression constructs of
the invention also encompass embodiments comprising allelic
variations of the human D4 dopamine receptor genomic DNA sequences
and cDNA-derived sequences. This invention includes recombinant
expression constructs comprising essentially the nucleotide
sequences of genomic and cDNA clones of the human D4 dopamine
receptor and allelic variations thereof in embodiments that provide
for the expression of human D4 dopamine receptor protein in
cultures of transformed eukaryotic cells.
It is also an object of this invention to provide cultures of
transformed eukayotic cells that have been transformed with such
recombinant expression constructs and that synthesize human D4
dopamine receptor protein. In a preferred embodiment, the invention
provides monkey COS cells that synthesize human D4 dopamine
receptor protein.
The present invention also includes protein preparations of the
human D4 dopamine receptor, and preparations of membranes
containing the human D4 dopamine receptor, derived from cultures of
eukaryotic cells transformed with the recombinant expression
constructs of the invention. In a preferred embodiment, cell
membranes containing human D4 dopamine receptor protein are
isolated from culture of COS-7 cells transformed with a recombinant
expression construct that directs the synthesis of human D4
dopamine receptor.
It also an object of this invention to provide the human D4
dopamine receptor for use in the in vitro screening of novel
antipsychotic compounds. In a preferred embodiment, membrane
preparations containing the human D4 dopamine receptor, derived
from cultures of eukaryotic cells transformed with the recombinant
expression constructs of the invention, are used to determine the
drug dissociation properties of antipsychotic compounds in vitro.
These properties are then used to characterize novel antipsychotic
compounds by comparison to the binding properties of known
antipsychotic compounds.
The present invention will also be useful for the detection of
dopamine and dopamine analogues, known or unknown, either naturally
occurring or as the embodiments of antipsychotic or other
drugs.
It is an object of the present invention to provide a method for
the quantitative detection of dopamine and dopamine analogues,
either naturally occurring or as the embodiments of antipsychotic
or other drugs. It is an additional object of the invention to
provide a method to detect dopamine or dopamine analogues in blood,
saliva, semen, cerebrospinal fluid, plasma, lymph, or any other
bodily fluid.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The term "D4 dopamine receptor" as used herein refers to proteins
substantially homologous to, and having substantially the same
biological activity as, the protein coded for by the nucleotide
sequences depicted in FIGS. 2A through 2D and FIGS. 6A through 6C
(i.e., proteins which display high affinity binding to clozapine)
[SEQ ID Nos: 1,3,4,5,7,8,10,12,14 & 15]. This definition is
intended to encompass natural allelic variations in the D4 dopamine
receptor sequence, specifically including the alleles D4.2, D4.4
and D4.7, as defined herein [SEQ ID Nos.: 17,19 & 21], and all
references to the D4 dopamine receptor, and nucleotide and amino
acid sequences thereof are intended to encompass such allelic
variations, both naturally-occurring and manmade. Cloned genes of
the present invention may code for D4 dopamine receptors of any
species of origin, including, mouse, rat, rabbit, cat, and human,
but preferably code for receptors of mammalian, most preferably
human, origin.
The production of proteins such as the D4 dopamine receptor from
cloned genes by genetic engineering is well known (see, e.g., U.S.
Pat. No. 4,761,371 to Bell et al. at Col. 6 line 3 to Col. 9 line
65; the disclosure of all U.S. patent references cited herein is to
be incorporated herein by reference). The discussion which follows
is accordingly intended as an overview of this field, and is not
intended to reflect the full state of the art.
DNA which encodes the D4 dopamine receptor may be obtained, in view
of the instant disclosure, by chemical synthesis, by screening
reverse transcripts of mRNA from appropriate tissues, cells or cell
line cultures, by screening genomic libraries from appropriate
cells, or by combinations of these procedures, as illustrated
below. Screening of mRNA or genomic DNA may be carried out with
oligonucleotide probes generated from the D4 dopamine receptor gene
sequence information provided herein. Probes may be labeled with a
detectable group such as a fluorescent group, a radioactive atom or
a chemiluminescent group in accordance with know procedures and
used in conventional hybridization assays, as described in greater
detail in the Examples below. In the alternative, D4 dopamine
receptor gene sequences may be obtained by use of the polymerase
chain reaction (PCR) procedure, with the PCR oligonucleotide
primers being produced from the D4 -dopamine receptor gene sequence
provided herein (see U.S. Pat. Nos. 4,683,195 to Mullis et al. and
4,683,202 to Mullis).
The D4 dopamine receptor may be synthesized in host cells
transformed with constructs containing DNA encoding the D4 dopamine
receptor. Such constructs are replicable and are used herein either
to amplify DNA encoding the D4 dopamine receptor and/or to express
DNA which encodes the D4 dopamine receptor. An expression construct
is a replicable DNA construct in which a DNA sequence encoding the
D4 receptor is operably linked to suitable control sequences
capable of effecting the expression of the D4 receptor in a
suitable host. The need for such control sequences will vary
depending upon the host selected and the transfection method
chosen. Generally, control sequences include a transcriptional
promoter, an optional operator sequence to control transcription, a
sequence encoding suitable mRNA ribosomal binding sites, and
sequences which control the termination of transcription and
translation. When used for DNA amplification such constructs do not
require expression control domains. All that is needed is the
ability to replicate in a host, usually conferred by an origin of
replication, and a selective marker gene to facilitate recognition
of transformants.
Constructs useful for practicing the present invention include
plasmids, viruses (including phage), retroviruses, and integratable
DNA fragments (i.e., fragments integratable into the host genome by
homologous recombination). The construct may replicate and function
independently of the host genome, or may, in some instances,
integrate into the host genome itself. Suitable constructs will
contain replicon and control sequences which are derived from
species compatible with the intended expression host. Transformed
host cells are cells which have been transformed, transfected or
infected with the D4 receptor-containing constructs assembled using
recombinant DNA techniques. Transformed host cells ordinarily
express the D4 receptor, but host cells transformed for purposes of
cloning or amplifying the D4 receptor DNA need not express the D4
receptor. When expressed, the D4 receptor will typically be located
in the host cell membrane.
DNA regions are operably linked when they are functionally related
to each other. For example: a promoter is operably linked to a
coding sequence if it controls the transcription of the sequence; a
ribosome binding site is operably linked to a coding sequence if it
is positioned so as to permit translation. Generally, operably
linked means contiguous and, in the case of leaders sequences,
contiguous and in the same translational reading frame.
Cultures of cells derived from multicellular organisms are a
desirable host for recombinant D4 dopamine receptor synthesis. In
principal, any higher eukaryotic cell culture can be used, whether
from vertebrate or invertebrate culture. However, mammalian cells
are preferred, as illustrated in the Examples. Propagation of such
cells in cell culture has become a routine procedure (see Tissue
Culture, Academic Press: New York (Kruse & Patterson, eds.)
1973). Examples of useful host cell lines are VERO and HeLa cells,
Chinese hamster ovary (CHO) cell lines, and WI138, BHK, COS-7, CV,
and MDCK cell lines. Expression constructs for such cells
ordinarily include (if necessary) an origin of replication, a
promoter located upstream from the gene to be expressed, along with
a ribosome binding site, RNA splice site (if intron-containing
genomic DNA is used), a polyadenylation site, and a transcriptional
termination sequence.
The transcriptional and translational control sequences in
expression constructs to be used in transforming vertebrate cells
are often provided by viral sources. For example, commonly used
promoters are derived from polyoma, Adenovirus 2, and Simian Virus
40 (SV40; see, e.g., U.S. Pat. No. 4,599,308). The early and late
promoters of SV40 are useful because both are obtained easily from
the virus within a fragment which also contains the SV40 viral
origin of replication (see Fiers et al., 1978, Nature 273: 113).
Further, the human genomic D4 receptor promoter, control and/or
signal sequences, may also be used, provided such control sequences
are compatible with the host cell chosen.
An origin of replication may be provided either within the
construct itself, such as may be derived from SV40 or other viral
source (e.g., Polyoma, Adenovirus, VSV, or MPV), or may be provided
by the host cell chromosomal replication mechanism. If the
construct is integrated into the host cell chromosome, the latter
may be sufficient.
D4 dopamine receptors made from cloned genes in accordance with the
present invention may be used for screening compounds for D4
dopamine receptor activity, or for determining the amount of a
dopaminergic drug in a solution (e.g., blood plasma or serum). For
example, host cells may be transformed with a construct of the
present invention, D4 dopamine receptors expressed in that host,
the cells lysed, and the membranes from those cells used to screen
compounds for D4 dopamine receptor binding activity. Competitive
binding assays in which such procedures may be carried out are well
known, as illustrated by the Examples below. By selection of host
cells which do not ordinarily express a dopamine receptor, pure
preparations of membranes containing D4 receptors can be obtained.
Further, D4 dopamine receptor agonist and antagonists can be
identified by transforming host cells with constructs of the
present invention. Membranes obtained from such cells can be used
in binding studies wherein the drug dissociation constants are
measured. Such cells must contain D4 protein in the plasma and
other cell membranes. Procedures for carrying out assays such as
these are also described in greater detail in the Examples which
follow.
Cloned genes and constructs of the present invention are useful to
transform cells which do not ordinarily express the D4 dopamine
receptor to thereafter express this receptor. Such cells are useful
as intermediates for making cell membrane preparations for receptor
binding assays, which are in turn useful for drug screening.
Further, genes and constructs of the present invention are useful
in gene therapy. For such purposes, retroviral constructs as
described in U.S. Pat. No. 4,650,764 to Temin and Watanabe or U.S.
Pat. No. 4,861,719 to Miller may be employed. Cloned genes of the
present invention, or fragments thereof, may also be used in gene
therapy carried out homologous recombination or site-directed
mutagenesis (See generally Thomas & Capecchi, 1987, Cell 51:
503-512; Bertling, 1987, Bioscience Reports 7: 107112; Smithies et
al., 1985, Nature 317: 230-234).
Cloned genes of the present invention, and oligonucleotides derived
therefrom, are useful for screening for restriction fragment length
polymorphism (RFLP) associated with genetic polymorphisms within a
population. Such RFLPs may also be associated with certain genetic
disorders, and the probes provided by the invention can be used for
their identification and the identification of individuals
susceptible to neuropsychiatric disorders such as schizophrenia and
manic depression. Such RFLPs may also be useful for predicting
individual responsiveness to psychotropic and antipsychotic
drugs.
Oligonucleotides of the present invention are useful as diagnostic
tools for probing D4 receptor gene expression in nervous tissue.
For example, tissue can be probed in situ with oligonucleotide
probes carrying detectable label groups by conventional
autoradiography techniques, as explained in greater detail in the
Examples below, to investigate native expression of this receptor
or pathological conditions relating thereto. Further, chromosomes
can be probed to investigate the location of the D4 dopamine
receptor gene, and potential pathological conditions related
thereto, as also illustrated by the Examples below.
Oligonueleotides of the present invention are also useful for in
vitro amplification of D4 dopamine receptor sequences.
Amplification methods include but are not intended to be limited to
the polymerase chain reaction and the ligase chain reaction.
Amplification of D4 dopamine receptor sequences is useful as a
diagnostic tools for analyzing and quantitating D4 receptor gene
expression in tissue, for example nervous tissue. Additionally, the
use of oligonucleotides synthesized or isolated according to
methods well known in the art that comprise D4 dopamine receptor
sequences provided by the invention permit in vitro amplification
methods to be used for the detection of D4 dopamine receptor
alleles comprising the genotype of somatic and germ cells, zygotes,
embryoes, and tissues in humans and other mammals for diagnostic,
therapeutic and other purposes.
The Examples which follow are illustrative of specific embodiments
of the invention, and various uses thereof. They are set forth for
explanatory purposes only, and are not to be taken as limiting the
invention.
EXAMPLE 1
Screening Tissue and Cell Line RNA for Dopamine Receptor
Expression
RNA was prepared from different rat tissues or cell lines using the
guadinium thiocyanate/CsCl procedure described in Bunzow et al.,
1988, Nature 336: 783-787. Tissues tested included heart,
epididymis, testis, gut, pancreas, spleen, thymus, muscle,
ventricle, atria, lung, adrenal, kidney, liver, pineal gland and
pituitary. Cell lines screened included SK-N-MC, SK-N-SH, COS,
AKR1, Ltk.sup.-, GH4C1, NG108-15, AtT20, 3T3, BSC40, C6, CV-1,
Hela, IMR-32, N4TG1, NCB-20, PC-12, Rin m5f and WERI-Rb-1. 20 .mu.g
of RNA was analyzed by Northern blot hybridization with a
radiolabeled BstYI-BglII DNA fragment of the rat D2 receptor, which
encodes the putative transmembrane domains VI and VII. Blots were
hybridized under standard conditions as described in Bunzow et al.,
ibid.; hybridization was performed overnight at 37.degree. C. Blots
were then washed at 55.degree. C. in 2.times. standard
saline-citrate (SSC) and 1% sodium dodecyl sulfate (SDS). Washed
blotes were exposed to X-ray film for two days at -70.degree.C.
using an intensifying screen. For comparison, the same blot was
hybridized under high stringency conditions (the modifications of
which include using 50% formamide and 42.degree. C. for the
hybridication and 0.2.times. SSC for the wash). Under conditions of
low stringency the SK-N-MC cell line showed a positive signal in
these experiments.
EXAMPLE 2
Construction of a cDNA Phage Library using Neuroblastoma RNA
Double-stranded cDNA was synthesized using standard techniques [see
Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2d
ed., Cold Spring Harbor Laboratory Press: New York] from
poly(A).sup.+ mRNA isolated from the human neuroblastoma cell line
SK-N-MC as described in Example 1. The cDNA was directionally
cloned into the EcoRI and XhoI restriction endonuclease sites of
the phage cloning vector lambda ZAPII (Stratagene, La Jolla,
Calif.). The library was transferred to colony plaque screen
filters (New England Nuclear, Boston, Mass.). Approximately 500,000
independent clones were screened under low-stringency hybridzation
conditions as described in Example 1. Hybridization was performed
for 30 hrs with .sup.32 P-labeled 1.6 kb BamHI-BglII and 300 bp
BstYI-BglII fragments of a rat D2 receptor clone at a specific
activity of 10.sup.6 dpm/.mu.g. Filters were washed at 55.degree.
C. in 2.times. SSC and 1% SDS. The clone D210S was isolated and
sequenced using the Sanger dideoxy chain termination method
catalyzed by Sequenase (U.S. Biochemical Corporation, Cleveland,
Ohio). The sequence of this clone is shown in FIG. 2 (hatched
area).
The putative coding sequence is shown in capitals (non-coding
sequence is in italics) and the deduced amino acid sequence is
shown above the nucleotide sequence. Numbering of the putative
coding sequence begins with the first methionine of the open
reading frame. The sequence corresponding to the cDNA clone is
hatched. Single-letter abbreviations for amino acids and
nucleotides used herein can be found in G. Zubay, Biochemistry (2d.
ed.), 1988 (MacMillen Publishing: New York) p.33. Noteworthy is the
presence of a duplicated 48 bp sequence in the putative third exon,
corresponding to the third cytoplamsic loop region of the D4
receptor protein. The complete nucleotide sequence of this clone
has been determined (see FIGS. 6A through 6C, wherein these
repeated sequences of this clone are designated D4.2 [SEQ ID No:
17]).
EXAMPLE 3
Screening a Genomic DNA Phage Library with a Human Dopamine
Receptor Probe
Clone D210S was .sup.32 P-labeled by random primed synthesis and
used to screen a commercially available human genomic library
cloned in the phage vector EMBL3 (Clonetech, Palo Alto, Cailf.).
Hybridization was performed as described in Example 2 using 50%
formamide. After hybridization the filters were washed at
65.degree. C. in 0.1.times. SSC and 0.1% SDS. The clone D210G was
isolated and analyzed by restriction endonuclease and Southern blot
analysis. The map of this genomic clone is shown in FIG. 1, wherein
the structure of the D4 receptor gene is compared with the
structure of the D2 gene. Relevant restriction endonuclease sites
in the D4 receptor sequence are indicated. The SalI site is part of
the cloning site in EMBL3. The proposed coding regions are boxed
and numbered in Roman numerals. Perfect matches of proposed
intron/exon junction sites are indicated by connecting stippled
bars between the receptor clones.
PstI-PstI fragments of approximately 1.3 kb and 2.6 kb, and an
overlapping SalI-EcoRI fragment of approximately 2.0 kb derived
from the D4 receptor gene were subcloned into the plasmid
pBluescript-SK (Stratagene). The subcloned fragments were
characterized by sequence analysis as described above. This
sequence is shown in FIGS. 2A through 2D. The complete nucleotide
sequence of this clone has been determined (see FIGS. 6A through
6C, wherein these repeated sequences of this clone are designated
D4.7 [SEQ ID No: 21]).
EXAMPLE 4
DNA Sequence Analysis of the Human D4 Dopamine Receptor
One of the cDNA clones detected by screening the SK-N-MC
neuroblastoma library with a rat D2 probe at low stringency (D210S)
contained a 780 bp EcoRI-XhoI insert which hybridized to the rat
probe. Screening of a human genomic EMBL3 library (Clontech) under
high stringency conditions with the clone D210S resulted in the
isolation of the genomic clone D210G.
Southern blot and sequence analysis indicated that the clone
contained a 5 kb SalI-PstI fragment which coded for the entire gene
of D210S [SEQ ID No.: 21]. Sequence analysis of this insert showed
the presence of an open reading frame with homology to the amino
acid sequence of transmembrane domains V (45%), VI (46%) and VII
(78%) of the D2 receptor, shown in FIGS. 3 and 3A through 3F. The
putative amino acid sequence of the human D4 receptor [SEQ ID No.:
22] is aligned with the human and rat D2, rat D3 and human and rat
D1 receptor sequences. Amino acids conserved within the group of
dopamine receptors are shaded. The putative transmembrane domains
are overlined and labeled by Roman numerals.
There is a potential translation initiation codon (ATG) 590 bp
downstream from the SalI site, followed by an open reading frame
that showed amino acid sequence homology with transmembrane domain
I (36%) and II (63%) of the D2 receptor. Almost immediately
downstream from the transmembrane domain II sequence, homology to
the D2 receptor disappears, indicating the presence of an intron in
the genomic DNA. This intron spanned approximately 2 kb, after
which sequence homology to the D2 receptor was re-established.
Translation of the putative gene product showed homology to the
transmembrane domains III (68%), IV (37%), V(46%) and VII (78%) of
the D2 receptor (see FIGS. 3 and 3A through 3F).
Potential splice junction donor and acceptor sites (Mount, 1982,
Nucl. Acids Res. 10: 461-472) were found in the transmembrane
domains II, III and VI, as shown in FIG. 1. These splice sites were
at an identical position as in the D2 and D3 receptor gene [see
Grandy et al., 1989, Proc. Nat. Acad. Sci. USA 86: 9762-9766; Dal
Toso et al., 1989, EMBO J. 8: 4025-4034; Sokoloff et al., 1990,
Nature 347: 146-151] and FIG. 1. The coding sequence downstream
from transmembrane domain IV is identical to the sequence of clone
D210S but is interrupted by an intron of about 300 bp between
transmembrane domains V and VI and an additional intron of 92 bp in
transmembrane VI (FIG. 1, hatched area). The precise location of
the splice site for the intron between transmembrane V and VI
cannot be determined due to the fact that a sequence of 52 bp
present in the coding sequence is repeated exactly on either side
of the intron (FIGS. 2A through 2D).
The deduced amino acid sequence from the genomic and cDNA
nucleotide sequences indicated that this gene codes for a protein
of 387 amino acids with an apparent molecular weight of 41 kD. A
hydrophobicity plot of the protein sequence suggests the existence
of seven transmembrane domains. These regions correlate with the
observed homologous regions in the human D2 receptor and other
receptors belonging to the family of G-protein coupled receptors
(Dohlman et al., 1987, Biochemistry 26: 2657-2664; Bunzow et al.,
1988, Nature 336: 783-787; Sokoloff et al., 1990, Nature 347:
146-151; and FIGS. 2A through 2D. A potential N-linked
glycosylation site (Hubbard & Ivatt, 1981, Ann. Rev. Biochem.
50: 555-583) is located two amino acids downstream from the
initiation methionine. The amino acid residues Asp (80) and Asp
(115) in the D4 receptor, which are conserved within the family
catecholaminergic receptors, are postulated to act as "counterions"
in catecholamine binding (Strader et al., 1988, J. Biol. Chem. 263:
10267-10271). Also conserved within the family of catecholaminergic
receptors are Ser (197) and Ser (700) which have been suggested to
interact with the catechol hydroxyl groups (Kozak, 1984, Nucleic
Acids Res. 12: 857-872). Several consensus sites for potential
phosphorylation by protein kinase C and protein kinase A are found
in the third cytoplasmic loop (Sibley et al., 1987, Cell 48:
913-922; Bouvier et al., 1988, Nature 333: 370-373). The Cys (187),
which may serve as a substrate for palmitoylation, is conserved in
most of the G-protein coupled receptors (O'Dowd et al., 1989, J.
Biol. Chem 264: 7564-7569). The short carboxyl tail, which
terminates similar to the D2 and D3 receptor at Cys (387) (Bunzow
et al., 1988, Nature 336: 783-787; Grandy et al., 1989, Proc. Natl.
Acad. Sci. USA 86: 9762-9766; Dal Toso et al., 1989, EMBO J. 8:
4025-4034; Sokoloff et al., 1990, Nature 347: 146-151), and the
relatively large third cytoplasmic loop, are features observed in
most receptors which interact with an isoform of the G protein.
A noteworthy feature of the sequence of the third exon of the
genomic D4 receptor clone is the presence of a 7-fold repeat of a
GC rich, 48 bp sequence, beginning at nucleotide 447 of exon III,
and encodes a proline-rich portion of the D4 dopamine receptor
protein (see FIGS. 6A through 6C, wherein these sequences of this
clone are designated D4.7 [SEQ ID No.:21]). This region of the
protein corresponds to the putative third cytoplasmic loop of the
receptor protein molecule [SEQ ID No.: 22]. This sequence
corresponds to the 2-fold repeat of a homologous sequence found in
the SK-N-MC neuroblastoma cDNA sequence described in Example 2,
suggesting that the D4 receptor gene may be polymorphic. This
sequence is uniquely found in the D4 receptor and is not homologous
to any other known dopamine receptor protein. Interestingly, this
region of the human D4 receptor is not found in the rat homologue
of the D4 receptor, making this variation specific to humans.
From these results we have concluded that the sequences we have
isolated encode a polymorphic member of the dopamine receptor
family.
EXAMPLE 5
Construction of an Mammalian DNA Expression Construct using
Dopamine Receptor cDNA
The ApaI-PstI gene fragment (FIG. 1, the PstI site found in exon
III after transmembrane domain V) was ligated to the corresponding
PstI-EcoRI cDNA fragment isolated from the SK-N-MC cDNA. This
construct was then cloned into the vector pCD-PS (Bonner et al.,
1988, Neuron 1: 403-410). This vector allows for the expression of
the human D4 receptor gene fom the SV40 promoter. Large quantities
of the pCD-PS-D4 construct plasmid were prepared using standard
techniques (see, Sambrook et al., ibid.). This plasmid was
transfected into COS-7 cells by the calcium phosphate precipitation
technique (Gorman et al., 1983, Science 221: 551-553). Two days
later membranes cells were harvested and analyzed as described in
Example 6.
EXAMPLE 6
Analysis of Dopamine and Dopamine-Antagonist Binding Of D4 Dopamine
Receptor
Cells were harvested and homogenized using a teflon pestle in 50 mM
Tris-HCl (pH 7.4 at 4.degree. C.) buffer containing 5 mM EDTA, 1.5
mM CaCl.sub.2, 5 mM MgCl.sub.2, 5 mM KCl and 120 mM NaCl.
Homogenates were centrifuged for 15 minutes at 39,000 g, and the
resulting pellets resuspended in buffer at a concentration of
150-250 .mu.g/ml. For saturation experiments, 0.25 ml aliquots of
each tissue homogenate was incubated in duplicate with increasing
concentrations of [.sup.3 H]spiperone (70.3 Ci/mmol; 10-3000 pM
final concentration) for 120 min at 22.degree. C. in a total volume
of 1 ml. The results of these experiments are shown in FIGS. 4A and
4B. The results shown are representative of two independent
experiments each conducted in duplicate (the inset show a Scatcherd
plot of the same data). Estimated B.sub.max (approximately 260
fmol/mg protein) and K.sub.i (70 pM) values were obtained by LIGAND
computer program.
Representative curves are shown in FIG. 5 for the concentration
dependent inhibition of [.sup.3 H]spiperone binding by various
dopaminergic agonist and antagonists. Estimated K.sub.i values are
listed in Table I along with the K.sub.i values obtained on the
human D2 receptor expressed in GH(4)ZR(7) cells. For competition
binding experiments, assays were initiated by the addition of 0.25
ml of membrane preparation and incubated in duplicate with the
concentrations of competing ligands indicated in FIG. 5 (10.sup.-14
to 10.sup.-3 M) and [.sup.3 H]spiperone (150-300 pM) for 120 min at
22.degree. C. Assays were terminated by rapid filtration through a
Titertek cell harvester and filters subsequently monitored to
quantitate radioactive tritium. For all experiments, specific
[.sup.3 H]spiperone binding was defined as that binding inhibited
by 10 .mu.M (+)sulpiride. Both saturation and competition binding
data were analyzed by the non-linear least square curve-fitting
program LIGAND run on a Digital Micro-PDP-11. The human D4 dopamine
receptor displays the following pharmacological profile of
inhibition of [.sup.3 H]spiperone binding in this assay:
spiperone>eticlopride>clozapine>(+)-butaclamol>raclopride>SCH23390.
EXAMPLE 7
Polymorphic Allelic Variants of the D4 Dopamine Receptor Isolated
from Human Tissue cDNA Libraries
Human cDNA libraries were screened for expression of polymorphic
variants of the human D4 receptor gene. A human substantia nigra
cDNA library constructed in lambda gt11 (Clontech) and a pituitary
cDNA library constructed in lambda gt10 as described in Example 2
were screened for clones encoding the D4 receptor. Approximately
0.5-1.times.10.sup.6 plaque-forming units (p.f.u.) were transferred
in duplicate to nylon filters (DuPont/NEN) and probed with a
.sup.32 P-labeled 700 bp EcoRI-XhoI fragment encoding the cDNA
isolated from the neuroepithelioma SK-N-MC under conditions as
described in Example 2 above.
Screening of eDNA libraries from human pituitary and substantia
nigra resulted in the isolation of variant eDNA clones of the D4
receptor. The pituitary lambda gt10 clone contained a 1.4-kb EcoRI
insert, coding for intron 1 and the down-stream sequences of the D4
receptor. This pituitary D4 receptor clone also contained the
second intron, but the last intron was spliced out. The isolated
substantia nigra lambda gt11 clone contained a 600-bp EcoRI insert,
coding for the D4 receptor, starting in the 5' site of the putative
third cytoplasmic loop. Both these clones contained a four-fold
repeat (see FIGS. 4A and 4B, wherein these sequences of these
clones are designated D4.4 [SEQ ID No.: 19]) of the 48-bp sequence
previously found as a 7-fold repeat in the D4 genomic clone D210G
(Example 4) and a 2-fold repeat in the neuroblastoma SK-N-MC cDNA
clone (Example 2) within the putative third cytoplasmic loop of the
D4 receptor protein (compare, SEQ ID Nos.: 18, 20 & 22]. A
comparison of the nucleic acid sequences revealed that, due to the
absence of conventional splice junction sites in the seven-fold
repeat sequence of the genomic clone, a novel splicing mechanism
would be required to account for the existence of the different
eDNA clones.
Two different human genomic libraries from different human
individuals (Clontech) were screened to detect allelic polymorphism
in the human D4 receptor gene. Screening of genomic libraries
resulted in the isolation of a genomic clone with a 4-fold repeat
of the 48 bp sequence previously detected in pituitary and
substantia nigra cDNA. This result indicated that the polymorphic
eDNA molecules resulted from genetic polymorphic variation in the
corresponding genomic DNA, due to the existence of polymorphic
alldes in the human population for the D4 receptor.
EXAMPLE 8
Additional D4 Receptor Gene Allelic Variants Found by RFLP Analysis
of Human Genomic DNA
The three different D4 receptor sequences predict a restriction
fragment length polymorphism for a HincII-PstI fragment of the D4
gene (FIGS. 6A through 6C). Southern blot analysis of human genomie
DNA was performed as described (see Sambrook et al., ibid. and
Example 3). A RFLP was observed in humans and the different alldie
fragments were sized.
Briefly, high molecular weight genomic DNA was isolated from human
blood samples using proteinase K and phenol/chloroform extractions.
Genomic DNA (5 .mu.g) was digested with the restriction
endonucleases HincII and PstI and size separated by agarose (1%)
gel electrophoresis. DNA was transferred to nylon membranes
(Zeta-probe, Biorad) according to standard techniques (Sambrook et
al., ibid.). Southern blots were probed with a .sup.32 P-labeled
600 bp EcoRI-HincII fragment, coding for the D4 cDNA isolated from
the neuroepithelioma SK-N-MC, and washed at high stringency
(65.degree. C., 0.1.times.SSC, 0.1% SDS, 40 min). The blot was
exposed to X-ray film for three days. Results of these experiments
are shown in FIG. 7.
The position of a 540-bp size marker is indicated on the left. D4
-hybridizing polymorphic bands can be seen at approximately 520 bp,
620 bp, 710 bp, 760 bp and 800 bp. [It will be recognized to those
with skill in this art that the sizes given herein for the alleles
of the human D4 dopamine receptor gene are limited in their
precision to the resolving power of the agarose gels used in the
analyses. The sizes are approximate as given herein, and more exact
sizes can be calculated from the sequences of the different alleles
found in SEQ ID Nos: 17, 19 & 21.] The 520 bp, 620 bp and 760
bp fragments correlate closely with the sizes of the HincII-PstI
fragments of the cloned D4 receptor variants with the two-, four-
and seven-fold repeat sequences respectively. The presence of 710
bp and 800 bp fragments suggests that variants with six-fold and
eight-fold repeat sequences also exist. Additional polulation
screening experiments have resulted in the detection of alleles
corresponding to three-fold and five-fold repeats. A total of 7
alleles of the D4 receptor gene have accordingly been found in the
human population.
EXAMPLE 9
Expression of Allelic Variants of the D4 Receptor
Mammalian DNA expression constructs were made as described in
Example 5 for expression of the allelic variants of the D4
receptor. Various cDNA constructs were cloned into the expression
vector pCD-PS (see Example 5) which contains the SV40 origin of
replication and drives expression of the cloned inserts from the
SV40 late promotor. A 1.7-kb KpnI-XbaI fragment comprising a eDNA
for the D4 receptor gene containing the 7-fold repeat was cloned
into the pCD-PS vector of Example 5 and called hereafter pCD-D4.7
(comprising the cDNA encoding the human D.sub.4 dopamine allele
identified as SEQ ID No.:21). Full-length cDNA clones for the D4.2
and D4.4 forms of the receptor were made by in vitro recombination
between partial cDNA clones of these forms with the full-length
eDNA clone of the D4.7 receptor variant. The clone pCD-D4.4
(comprising the cDNA encoding the human D.sub.4 dopamine allele
identified as SEQ ID No.:19) was created by substituting the 920-bp
PstI-EcoRI 3' fragment of pCD-D4.7 with the 730-bp PstI-EcoRI
fragment of the D4 cDNA isolated from human pituitary. In a similar
fashion the clone pCD-D4.2 (comprising the cDNA encoding the human
D.sub.4 dopamine allele identified as SEQ ID No.:17) was
constructed by exchange of this 3' PstI-EcoRI fragment of pCD-D4.7
with a 630-bp PstI-EcoRI fragment of the D4.2 cDNA clone isolated
from the neuroepithelioma SK-N-MC.
Transient expression in COS-7 cells was achieved as follows. Cells
harvested and washed in phosphate buffered saline (PBS).
5.times.10.sup.7 cells were resuspended in 1 ml PBS with 100
.mu.g/ml plasmid DNA (purified by caesium chloride gradient
centrifugation) and incubated for 10 rain on ice. Next, 400 .mu.l
aliquots of the cell suspension were subjected to an electric field
of 0.65 kV/cm, 4.1 ms pulse duration using a BTX 600 Electro Cell
Manipulator (Biotechnologies & Experimental Research, Inc., San
Diego, Calif.). After the electric pulse, the cells were incubated
for another 10 min on ice and then seeded in Modified Eagle's
Medium supplemented with 10% fetal calf serum. The next day the
medium was renewed. Three days after electroporation the cells were
harvested and stored at -80.degree. C. until use in receptor
binding studies as described herein
Expression of each of the cloned variant D4 receptor constructs was
demonstrated by Northern blot analysis as described in Example 1.
Blots were hybridized with the 700 bp EcoRI-XhoI fragment of the D4
cDNA isolated from the neuroepithelioma SK-N-MC (Example 2). The
results of these experiments are shown in FIG. 8. Transient
expression of the three forms in COS-7 cells as characterized in
these experiments demonstrated the expected size and size
differences between the three forms, indicating that none of the
expressed D4 receptor RNAs are further processed or produced from
one another by RNA splicing events. Furthermore, the two bands
observed for the D4.2 and D4.4 clones represent the consequence of
the use of either the endogenous D4 receptor polyadenylation signal
or the SV40 (vector-derived) polyadenylation signal). These
observations indicate that in the transient expression system the
expression of the three different clones would result in the
formation of three structurally different receptors.
EXAMPLE 10
Analysis of Dopamine and Dopamine-Antagonist Binding of Variant D4
Dopamine Receptors
Pharmacological analysis of dopamine agonist and antagonist binding
was performed as described in Example 6. The results of these
experiments are shown in FIGS. 9a and 9b. FIG. 9a illustrate
Scatchard analysis of the saturation isotherms for [.sup.3
H]spiperone binding to membranes prepared from COS-7 cells
transiently transfected with pCD-D4.2 (comprising the cDNA encoding
the human D.sub.4 dopamine allele identified as SEQ ID No.:17),
pCD-D4.4 (comprising the cDNA encoding the human D.sub.4 dopamine
allele identified as SEQ ID No.:19) and pCD-D4.7 (comprising the
cDNA encoding the human D.sub.4 dopamine allele identified as SEQ
ID No.:21). FIG. 9b show clozapine competition of [.sup.3
H]spiperone binding for the three alldie forms of the D4 receptor
in the presence (+Na.sup.+) and absence (-Na.sup.+) of sodium
chloride.
Pharmacological analysis demonstrated that all three variants
displayed saturable [.sup.3 H]spiperone binding (300-1000 fmol
mg.sup.-1) with similar dissociation constants in the absence of
sodium chloride (K.sub.d =40-50 pM; FIG. 4A). However, in the
presence of 120 mM sodium chloride, the dissociation constants
increased approximately two- to three-fold for D4.2 and D4.4 but
not for D4.7.
Clozapine competition of [.sup.3 H]spiperone binding revealed that
D4.2 and D4.4 had lower dissociation constants for clozapine in the
absence of sodium chloride (K.sub.i =3 nM without sodium chloride;
K.sub.i =23 nM with sodium chloride). D4.7 had a dissociation
constant of approximately 15 nM for clozapine which did not exhibit
sodium chloride sensitivity (K.sub.i =12 nM without sodium
chloride; K.sub.i =18 nM with sodium chloride; shown in FIG. 4B).
This sodium chloride-mediated effect for clozapine on the D4
variants was not modulated by guanine nucleotides.
Agonists and antagonists (dopamine, bromocriptine, ratiopride and
clozapine) inhibited [.sup.3 H]spiperone binding (in the presence
of sodium chloride) to these different D4 receptor variants in a
concentration-dependent manner with similar dissociations
constants. Furthermore, all three variants exhibited a guanine
nucleotide-sensitive high-affinity form of the receptor upon
competition with dopamine, suggesting that all these variants can
functionally couple to G-proteins. Thus, we have defined a novel,
polymorphic dopamine receptor which we term D4.
It should be understood that the foregoing disclosure emphasizes
certain specific embodiments of the invention and that all
modifications or alternatives equivalent thereto are within the
spirit and scope of the invention as set forth in the appended
claims.
__________________________________________________________________________
SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF
SEQUENCES: 22 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 388 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
DNA (genomic) (ix) FEATURE: (A) NAME/KEY: 5'UTR (B) LOCATION:
1..103 (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION: 104..388 (ix)
FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 104..388 (x) PUBLICATION
INFORMATION: (A) AUTHORS: Van Tol, Hubert H.M. Wu, Caren M. Guan,
Hong- Chang Ohara, Koichi Bunzow, James R. Civelli, Olivier
Kennedy, James Seeman, Phillip Niznik, Hyman B. Jovanovic, Vera (B)
TITLE: Multiple dopamine D4 receptor variants in the human
population (C) JOURNAL: Nature (D) VOLUME: 358 (F) PAGES: 149-152
(G) DATE: 9 JULY-1992 (x) PUBLICATION INFORMATION: (A) AUTHORS: Van
Tol, Hubert H.M. Bunzow, James R. Guan, Hong- Chang Sunahara, Roger
K. Seeman, Phillip Niznik, Hyman B. Civelli, Olivier (B) TITLE:
Cloning of the gene for a human dopamine D4 receptor with high
affinity for the antipsychotic clozapine (C) JOURNAL: Nature (D)
VOLUME: 350 (F) PAGES: 610-614 (G) DATE: 18 April-1991 (K) RELEVANT
RESIDUES IN SEQ ID NO:1: FROM 1 TO 388 (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:1:
CGGGGGCGGGACCAGGGTCCGGCCGGGGCGTGCCCCCGGGGAGGGACTCCCCGGCTTGCC60
CCCCGGCGTTGTCCGCGGTGCTCAGCGCCCGCCCGGGCGCGCCATGGGGAACCGC115
MetGlyAsnArg AGCACCGCGGACGCGGACGGGCTGCTGGCTGGGCGCGGGCGGGCCGCG163
SerThrAlaAspAlaAspGlyLeuLeuAlaGlyArgGlyArgAlaAla 5101520
GGGGCATCTGCGGGGGCATCTGCGGGGCTGGCTGGGCAGGGCGCGGCG211
GlyAlaSerAlaGlyAlaSerAlaGlyLeuAlaGlyGlnGlyAlaAla 253035
GCGCTGGTGGGGGGCGTGCTGCTCATCGGCGCGGTGCTCGCGGGGAAC259
AlaLeuValGlyGlyValLeuLeuIleGlyAlaValLeuAlaGlyAsn 404550
TCGCTCGTGTGCGTGAGCGTGGCCACCGAGCGCGCCCTGCAGACGCCC307
SerLeuValCysValSerValAlaThrGluArgAlaLeuGlnThrPro 556065
ACCAACTCCTTCATCGTGAGCCTGGCGGCCGCCGACCTCCTCCTCGCT355
ThrAsnSerPheIleValSerLeuAlaAlaAlaAspLeuLeuLeuAla 707580
CTCCTGGTGCTGCCGCTCTTCGTCTACTCCGAG388
LeuLeuValLeuProLeuPheValTyrSerGlu 859095 (2) INFORMATION FOR SEQ ID
NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 95 amino acids (B)
TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
MetGlyAsnArgSerThrAlaAspAlaAspGlyLeuLeuAlaGlyArg 151015
GlyArgAlaAlaGlyAlaSerAlaGlyAlaSerAlaGlyLeuAlaGly 202530
GlnGlyAlaAlaAlaLeuValGlyGlyValLeuLeuIleGlyAlaVal 354045
LeuAlaGlyAsnSerLeuValCysValSerValAlaThrGluArgAla 505560
LeuGlnThrProThrAsnSerPheIleValSerLeuAlaAlaAlaAsp 65707580
LeuLeuLeuAlaLeuLeuValLeuProLeuPheValTyrSerGlu 859095 (2)
INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix)
FEATURE: (A) NAME/KEY: intron (B) LOCATION: 1..20 (C)
IDENTIFICATION METHOD: experimental (D) OTHER INFORMATION: /partial
/cons.sub.-- splice=(5'site: YES, 3'site: NO)
/evidence=EXPERIMENTAL /label=IntronI /note="This is the 5'sequence
of an intron estimated to be 2.0 kilobases in length" (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:3: GTGAGCCGCGTCCGGCCGCA20 (2) INFORMATION
FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: (A)
NAME/KEY: intron (B) LOCATION: 1..20 (C) IDENTIFICATION METHOD:
experimental (D) OTHER INFORMATION: /partial /cons.sub.--
splice=(5'site: NO, 3'site: YES) /evidence=EXPERIMENTAL
/label=IntronI /note="This is the 3'sequence of a intron estimated
to be 2.0 kilobases in length." (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:4: CCTGTGGTGTCGCCGCGCAG20 (2) INFORMATION FOR SEQ ID NO:5: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 113 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (ix) FEATURE: (A) NAME/KEY: exon (B)
LOCATION: 1..113 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:
1..113 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GTCCAGGGTGGCGCGTGGCTGCTGAGCCCCCGCCTGTGCGACGCCCTC48
ValGlnGlyGlyAlaTrpLeuLeuSerProArgLeuCysAspAlaLeu 151015
ATGGCCATGGACGTCATGCTGTGCACCGCCTCCATCTTCAACCTGTGC96
MetAlaMetAspValMetLeuCysThrAlaSerIlePheAsnLeuCys 202530
GCCATCAGCGTGGACAG113 AlaIleSerValAsp 35 (2) INFORMATION FOR SEQ ID
NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 amino acids (B)
TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
ValGlnGlyGlyAlaTrpLeuLeuSerProArgLeuCysAspAlaLeu 151015
MetAlaMetAspValMetLeuCysThrAlaSerIlePheAsnLeuCys 202530
AlaIleSerValAsp 35 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 102 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
DNA (genomic) (ix) FEATURE: (A) NAME/KEY: intron (B) LOCATION:
1..102 (C) IDENTIFICATION METHOD: experimental (D) OTHER
INFORMATION: /evidence=EXPERIMENTAL /label=IntronII (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:7:
GTGCGCCGCCCTCCCCGCCCGCGCCCCGGCGCCCCCGCGCCCCGCCCGCCGCCCTCACCG60
CGGCCTGTGCGCTGTCCGGCGCCCCCTCGGCGCTCCCCGCAG102 (2) INFORMATION FOR
SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 563 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: (A)
NAME/KEY: exon (B) LOCATION: 1..563 (C) IDENTIFICATION METHOD:
experimental (D) OTHER INFORMATION: /evidence=EXPERIMENTAL
/standard.sub.-- name="Alternate Exon 3: D4.2" /note="This sequence
represent the sequence of the third exon of allele D4.2 of the
human D4 dopamine receptor gene" (ix) FEATURE: (A) NAME/KEY:
misc.sub.-- feature (B) LOCATION: 257..262 (C) IDENTIFICATION
METHOD: experimental (D) OTHER INFORMATION: /function="Polymorphic
PstI site" /evidence=EXPERIMENTAL /label=PstI /note="This feature
is the site of one of the restriction enzymes whereby digestion of
genomic DNA produces a RFLP " (ix) FEATURE: (A) NAME/KEY:
repeat.sub.-- region (B) LOCATION: 346..442 (D) OTHER INFORMATION:
/rpt.sub.-- type="tandem" /rpt.sub.-- unit=348 .. 396 /note="This
sequence represents one of 7 known alleles of human D4 dopamine
receptor gene encoding a 16 amino acid sequence repeated twice (ix)
FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 2..563 (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:8:
GTTCGTGGCCGTGGCCGTGCCGCTGCGCTACAACCGGCAGGGTGGG46
PheValAlaValAlaValProLeuArgTyrAsnArgGlnGlyGly 151015
AGCCGCCGGCAGCTGCTGCTCATCGGCGCCACGTGGCTGCTGTCCGCG94
SerArgArgGlnLeuLeuLeuIleGlyAlaThrTrpLeuLeuSerAla 202530
GCGGTGGCGGCGCCCGTACTGTGCGGCCTCAACGACGTGCGCGGCCGC142
AlaValAlaAlaProValLeuCysGlyLeuAsnAspValArgGlyArg 354045
GACCCCGCCGTGTGCCGCCTGGAGGACCGCGACTACGTGGTCTACTCG190
AspProAlaValCysArgLeuGluAspArgAspTyrValValTyrSer 505560
TCCGTGTGCTCCTTCTTCCTACCCTGCCCGCTCATGCTGCTGCTGTAC238
SerValCysSerPhePheLeuProCysProLeuMetLeuLeuLeuTyr 657075
TGGGCCACGTTCCGCGGCCTGCAGCGCTGGGAGGTGGCACGTCGCGCC286
TrpAlaThrPheArgGlyLeuGlnArgTrpGluValAlaArgArgAla 80859095
AAGCTGCACGGCCGCGCGCCCCGCCGACCCAGCGGCCCTGGCCCGCCT334
LysLeuHisGlyArgAlaProArgArgProSerGlyProGlyProPro 100105110
TCCCCCACGCCACCCGCGCCCCGCCTCCCCCAGGACCCCTGCGGCCCC382
SerProThrProProAlaProArgLeuProGlnAspProCysGlyPro 115120125
GACTGTGCGCCCCCCGCGCCCGGCCTCCCCCCGGACCCCTGCGGCTCC430
AspCysAlaProProAlaProGlyLeuProProAspProCysGlySer 130135140
AACTGTGCTCCCCCCGACGCCGTCAGAGCCGCCGCGCTCCCACCCCAG478
AsnCysAlaProProAspAlaValArgAlaAlaAlaLeuProProGln
145150155 ACTCCACCGCAGACCCGCAGGAGGCGGCGTGCCAAGATCACCGGCCGG526
ThrProProGlnThrArgArgArgArgArgAlaLysIleThrGlyArg 160165170175
GAGCGCAAGGCCATGAGGGTCCTGCCGGTGGTGGTCG563
GluArgLysAlaMetArgValLeuProValValVal 180185 (2) INFORMATION FOR SEQ
ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 187 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
PheValAlaValAlaValProLeuArgTyrAsnArgGlnGlyGlySer 151015
ArgArgGlnLeuLeuLeuIleGlyAlaThrTrpLeuLeuSerAlaAla 202530
ValAlaAlaProValLeuCysGlyLeuAsnAspValArgGlyArgAsp 354045
ProAlaValCysArgLeuGluAspArgAspTyrValValTyrSerSer 505560
ValCysSerPhePheLeuProCysProLeuMetLeuLeuLeuTyrTrp 65707580
AlaThrPheArgGlyLeuGlnArgTrpGluValAlaArgArgAlaLys 859095
LeuHisGlyArgAlaProArgArgProSerGlyProGlyProProSer 100105110
ProThrProProAlaProArgLeuProGlnAspProCysGlyProAsp 115120125
CysAlaProProAlaProGlyLeuProProAspProCysGlySerAsn 130135140
CysAlaProProAspAlaValArgAlaAlaAlaLeuProProGlnThr 145150155160
ProProGlnThrArgArgArgArgArgAlaLysIleThrGlyArgGlu 165170175
ArgLysAlaMetArgValLeuProValValVal 180185 (2) INFORMATION FOR SEQ ID
NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 659 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: (A) NAME/KEY: exon
(B) LOCATION: 1..659 (C) IDENTIFICATION METHOD: experimental (D)
OTHER INFORMATION: /evidence=EXPERIMENTAL /standard.sub.--
name="Alternate Exon 3: D4.4" /note="This sequence represents the
third exon of allele D4.4 of the human D4 dopamine receptor gene"
(ix) FEATURE: (A) NAME/KEY: misc.sub.-- feature (B) LOCATION:
257..262 (C) IDENTIFICATION METHOD: experimental (D) OTHER
INFORMATION: /function="PstI site" /evidence=EXPERIMENTAL
/standard.sub.-- name="PstI site" /label=PstI /note="This sequence
represents a polymorphic PstI site whereby digestion of human
genomic DNA produces a RFLP " (ix) FEATURE: (A) NAME/KEY:
repeat.sub.-- region (B) LOCATION: 346..538 (C) IDENTIFICATION
METHOD: experimental (D) OTHER INFORMATION: /rpt.sub.--
type="tandem" /evidence=EXPERIMENTAL /rpt.sub.-- unit=348 .. 396
/note="This repeat is present in 7 known alleles of the human D4
dopamine receptor gene and encodes a 16 amino acid sequence
repeated 4 times in the (ix) FEATURE: (A) NAME/KEY: CDS (B)
LOCATION: 2..659 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GTTCGTGGCCGTGGCCGTGCCGCTGCGCTACAACCGGCAGGGTGGG46
PheValAlaValAlaValProLeuArgTyrAsnArgGlnGlyGly 151015
AGCCGCCGGCAGCTGCTGCTCATCGGCGCCACGTGGCTGCTGTCCGCG94
SerArgArgGlnLeuLeuLeuIleGlyAlaThrTrpLeuLeuSerAla 202530
GCGGTGGCGGCGCCCGTACTGTGCGGCCTCAACGACGTGCGCGGCCGC142
AlaValAlaAlaProValLeuCysGlyLeuAsnAspValArgGlyArg 354045
GACCCCGCCGTGTGCCGCCTGGAGGACCGCGACTACGTGGTCTACTCG190
AspProAlaValCysArgLeuGluAspArgAspTyrValValTyrSer 505560
TCCGTGTGCTCCTTCTTCCTACCCTGCCCGCTCATGCTGCTGCTGTAC238
SerValCysSerPhePheLeuProCysProLeuMetLeuLeuLeuTyr 657075
TGGGCCACGTTCCGCGGCCTGCAGCGCTGGGAGGTGGCACGTCGCGCC286
TrpAlaThrPheArgGlyLeuGlnArgTrpGluValAlaArgArgAla 80859095
AAGCTGCACGGCCGCGCGCCCCGCCGACCCAGCGGCCCTGGCCCGCCT334
LysLeuHisGlyArgAlaProArgArgProSerGlyProGlyProPro 100105110
TCCCCCACGCCACCCGCGCCCCGCCTCCCCCAGGACCCCTGCGGCCCC382
SerProThrProProAlaProArgLeuProGlnAspProCysGlyPro 115120125
GACTGTGCGCCCCCCGCGCCCGGCCTTCCCCGGGGTCCCTGCGGCCCC430
AspCysAlaProProAlaProGlyLeuProArgGlyProCysGlyPro 130135140
GACTGTGCGCCCGCCGCGCCCAGCCTCCCCCAGGACCCCTGCGGCCCC478
AspCysAlaProAlaAlaProSerLeuProGlnAspProCysGlyPro 145150155
GACTGTGCGCCCCCCGCGCCCGGCCTCCCCCCGGACCCCTGCGGCTCC526
AspCysAlaProProAlaProGlyLeuProProAspProCysGlySer 160165170175
AACTGTGCTCCCCCCGACGCCGTCAGAGCCGCCGCGCTCCCACCCCAG574
AsnCysAlaProProAspAlaValArgAlaAlaAlaLeuProProGln 180185190
ACTCCACCGCAGACCCGCAGGAGGCGGCGTGCCAAGATCACCGGCCGG622
ThrProProGlnThrArgArgArgArgArgAlaLysIleThrGlyArg 195200205
GAGCGCAAGGCCATGAGGGTCCTGCCGGTGGTGGTCG659
GluArgLysAlaMetArgValLeuProValValVal 210215 (2) INFORMATION FOR SEQ
ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 219 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
PheValAlaValAlaValProLeuArgTyrAsnArgGlnGlyGlySer 151015
ArgArgGlnLeuLeuLeuIleGlyAlaThrTrpLeuLeuSerAlaAla 202530
ValAlaAlaProValLeuCysGlyLeuAsnAspValArgGlyArgAsp 354045
ProAlaValCysArgLeuGluAspArgAspTyrValValTyrSerSer 505560
ValCysSerPhePheLeuProCysProLeuMetLeuLeuLeuTyrTrp 65707580
AlaThrPheArgGlyLeuGlnArgTrpGluValAlaArgArgAlaLys 859095
LeuHisGlyArgAlaProArgArgProSerGlyProGlyProProSer 100105110
ProThrProProAlaProArgLeuProGlnAspProCysGlyProAsp 115120125
CysAlaProProAlaProGlyLeuProArgGlyProCysGlyProAsp 130135140
CysAlaProAlaAlaProSerLeuProGlnAspProCysGlyProAsp 145150155160
CysAlaProProAlaProGlyLeuProProAspProCysGlySerAsn 165170175
CysAlaProProAspAlaValArgAlaAlaAlaLeuProProGlnThr 180185190
ProProGlnThrArgArgArgArgArgAlaLysIleThrGlyArgGlu 195200205
ArgLysAlaMetArgValLeuProValValVal 210215 (2) INFORMATION FOR SEQ ID
NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 803 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: (A) NAME/KEY: exon
(B) LOCATION: 1..803 (C) IDENTIFICATION METHOD: experimental (D)
OTHER INFORMATION: /evidence=EXPERIMENTAL /standard.sub.--
name="Alternate Exon 3: D4.7" /note="This sequence represents the
third exon of allele D4.7 of the human D4 dopamine receptor gene"
(ix) FEATURE: (A) NAME/KEY: misc.sub.-- feature (B) LOCATION:
257..262 (C) IDENTIFICATION METHOD: experimental (D) OTHER
INFORMATION: /function="PstI site" /evidence=EXPERIMENTAL
/standard.sub.-- name="PstI site" /label=PstI /note="This sequence
is a PstI site whereby digestion of human genomic DNA produces a
RFLP" (ix) FEATURE: (A) NAME/KEY: repeat.sub.-- region (B)
LOCATION: 346..682 (C) IDENTIFICATION METHOD: experimental (D)
OTHER INFORMATION: /rpt.sub.-- type="tandem" /evidence=EXPERIMENTAL
/rpt.sub.-- unit=346 .. 394 /note="This sequence is a repeat found
in 7 known alleles of the human D4 dopamine receptor gene encoding
a 16 amino acid sequence repeated 7 times (ix) FEATURE: (A)
NAME/KEY: CDS (B) LOCATION: 2..803 (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:12: GTTCGTGGCCGTGGCCGTGCCGCTGCGCTACAACCGGCAGGGTGGG46
PheValAlaValAlaValProLeuArgTyrAsnArgGlnGlyGly 151015
AGCCGCCGGCAGCTGCTGCTCATCGGCGCCACGTGGCTGCTGTCCGCG94
SerArgArgGlnLeuLeuLeuIleGlyAlaThrTrpLeuLeuSerAla 202530
GCGGTGGCGGCGCCCGTACTGTGCGGCCTCAACGACGTGCGCGGCCGC142
AlaValAlaAlaProValLeuCysGlyLeuAsnAspValArgGlyArg 354045
GACCCCGCCGTGTGCCGCCTGGAGGACCGCGACTACGTGGTCTACTCG190
AspProAlaValCysArgLeuGluAspArgAspTyrValValTyrSer 505560
TCCGTGTGCTCCTTCTTCCTACCCTGCCCGCTCATGCTGCTGCTGTAC238
SerValCysSerPhePheLeuProCysProLeuMetLeuLeuLeuTyr 657075
TGGGCCACGTTCCGCGGCCTGCAGCGCTGGGAGGTGGCACGTCGCGCC286
TrpAlaThrPheArgGlyLeuGlnArgTrpGluValAlaArgArgAla 80859095
AAGCTGCACGGCCGCGCGCCCCGCCGACCCAGCGGCCCTGGCCCGCCT334
LysLeuHisGlyArgAlaProArgArgProSerGlyProGlyProPro 100105110
TCCCCCACGCCACCCGCGCCCCGCCTCCCCCAGGACCCCTGCGGCCCC382
SerProThrProProAlaProArgLeuProGlnAspProCysGlyPro 115120125
GACTGTGCGCCCCCCGCGCCCGGCCTTCCCCGGGGTCCCTGCGGCCCC430
AspCysAlaProProAlaProGlyLeuProArgGlyProCysGlyPro 130135140
GACTGTGCGCCCGCCGCGCCCGGCCTCCCCCCGGACCCCTGCGGCCCC478
AspCysAlaProAlaAlaProGlyLeuProProAspProCysGlyPro 145150155
GACTGTGCGCCCCCCGCGCCCGGCCTCCCCCAGGACCCCTGCGGCCCC526
AspCysAlaProProAlaProGlyLeuProGlnAspProCysGlyPro 160165170175
GACTGTGCGCCCCCCGCGCCCGGCCTTCCCCGGGGTCCCTGCGGCCCC574
AspCysAlaProProAlaProGlyLeuProArgGlyProCysGlyPro 180185190
GACTGTGCGCCCCCCGCGCCCGGCCTCCCCCAGGACCCCTGCGGCCCC622
AspCysAlaProProAlaProGlyLeuProGlnAspProCysGlyPro 195200205
GACTGTGCGCCCCCCGCGCCCGGCCTCCCCCCGGACCCCTGCGGCTCC670
AspCysAlaProProAlaProGlyLeuProProAspProCysGlySer 210215220
AACTGTGCTCCCCCCGACGCCGTCAGAGCCGCCGCGCTCCCACCCCAG718
AsnCysAlaProProAspAlaValArgAlaAlaAlaLeuProProGln 225230235
ACTCCACCGCAGACCCGCAGGAGGCGGCGTGCCAAGATCACCGGCCGG766
ThrProProGlnThrArgArgArgArgArgAlaLysIleThrGlyArg 240245250255
GAGCGCAAGGCCATGAGGGTCCTGCCGGTGGTGGTCG803
GluArgLysAlaMetArgValLeuProValValVal 260265 (2) INFORMATION FOR SEQ
ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 267 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:13:
PheValAlaValAlaValProLeuArgTyrAsnArgGlnGlyGlySer 151015
ArgArgGlnLeuLeuLeuIleGlyAlaThrTrpLeuLeuSerAlaAla 202530
ValAlaAlaProValLeuCysGlyLeuAsnAspValArgGlyArgAsp 354045
ProAlaValCysArgLeuGluAspArgAspTyrValValTyrSerSer 505560
ValCysSerPhePheLeuProCysProLeuMetLeuLeuLeuTyrTrp 65707580
AlaThrPheArgGlyLeuGlnArgTrpGluValAlaArgArgAlaLys 859095
LeuHisGlyArgAlaProArgArgProSerGlyProGlyProProSer 100105110
ProThrProProAlaProArgLeuProGlnAspProCysGlyProAsp 115120125
CysAlaProProAlaProGlyLeuProArgGlyProCysGlyProAsp 130135140
CysAlaProAlaAlaProGlyLeuProProAspProCysGlyProAsp 145150155160
CysAlaProProAlaProGlyLeuProGlnAspProCysGlyProAsp 165170175
CysAlaProProAlaProGlyLeuProArgGlyProCysGlyProAsp 180185190
CysAlaProProAlaProGlyLeuProGlnAspProCysGlyProAsp 195200205
CysAlaProProAlaProGlyLeuProProAspProCysGlySerAsn 210215220
CysAlaProProAspAlaValArgAlaAlaAlaLeuProProGlnThr 225230235240
ProProGlnThrArgArgArgArgArgAlaLysIleThrGlyArgGlu 245250255
ArgLysAlaMetArgValLeuProValValVal 260265 (2) INFORMATION FOR SEQ ID
NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 94 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: (A) NAME/KEY:
intron (B) LOCATION: 1..94 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
GTGGGTTCCTGTCCTGAGGGGCGGGGAGGAGAGGAGGGGGGGAGTACGAGGCCGGCTGGG60
CGGGGGGCGCTAACGCGGCTCTCGGCGCCCCCAG94 (2) INFORMATION FOR SEQ ID
NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 328 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: (A) NAME/KEY: exon
(B) LOCATION: 1..328 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:
3..203 (ix) FEATURE: (A) NAME/KEY: 3'UTR (B) LOCATION: 204..328
(ix) FEATURE: (A) NAME/KEY: polyA.sub.-- site (B) LOCATION: 304
(ix) FEATURE: (A) NAME/KEY: misc.sub.-- feature (B) LOCATION:
36..41 (C) IDENTIFICATION METHOD: experimental (D) OTHER
INFORMATION: /function="HinCII site" /evidence=EXPERIMENTAL
/standard.sub.-- name="HinCII site" /label=HinCII /note="This
sequence is a HinCII site whereby digestion of genomic DNA produces
a RFLP" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
GGGCCTTCCTGCTGTGCTGGACGCCCTTCTTCGTGGTGCACATCACG47
AlaPheLeuLeuCysTrpThrProPhePheValValHisIleThr 151015
CAGGCGCTGTGTCCTGCCTGCTCCGTGCCCCCGCGGCTGGTCAGCGCC95
GlnAlaLeuCysProAlaCysSerValProProArgLeuValSerAla 202530
GTCACCTGGCTGGGCTACGTCAACAGCGCCCTCACCCCCGTCATCTAC143
ValThrTrpLeuGlyTyrValAsnSerAlaLeuThrProValIleTyr 354045
ACTGTCTTCAACGCCGAGTTCCGCAACGTCTTCCGCAAGGCCCTGCGT191
ThrValPheAsnAlaGluPheArgAsnValPheArgLysAlaLeuArg 505560
GCCTGCTGCTGAGCCGGGCACCCCCGGACGCCCCCCGGCCTGATGGCCA240 AlaCysCys 65
GGCCTCAGGGACCAAGGAGATGGGGAGGGCGCTTTTGTACGTTAATTAAACAAATTCCTT300
CCCAAACTCAGCTGTGAAGGCTCCTGGG328 (2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 66 amino acids (B) TYPE:
amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:16:
AlaPheLeuLeuCysTrpThrProPhePheValValHisIleThrGln 151015
AlaLeuCysProAlaCysSerValProProArgLeuValSerAlaVal 202530
ThrTrpLeuGlyTyrValAsnSerAlaLeuThrProValIleTyrThr 354045
ValPheAsnAlaGluPheArgAsnValPheArgLysAlaLeuArgAla 505560 CysCys 65
(2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 1370 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE:
(A) NAME/KEY: 5'UTR (B) LOCATION: 1..103 (ix) FEATURE: (A)
NAME/KEY: 3'UTR (B) LOCATION: 1268..1370 (ix) FEATURE: (A)
NAME/KEY: CDS (B) LOCATION: 104..1267 (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:17:
CGGGGGCGGGACCAGGGTCCGGCCGGGGCGTGCCCCCGGGGAGGGACTCCCCGGCTTGCC60
CCCCGGCGTTGTCCGCGGTGCTCAGCGCCCGCCCGGGCGCGCCATGGGGAACCGC115
MetGlyAsnArg 1 AGCACCGCGGACGCGGACGGGCTGCTGGCTGGGCGCGGGCGGGCCGCG163
SerThrAlaAspAlaAspGlyLeuLeuAlaGlyArgGlyArgAlaAla 5101520
GGGGCATCTGCGGGGGCATCTGCGGGGCTGGCTGGGCAGGGCGCGGCG211
GlyAlaSerAlaGlyAlaSerAlaGlyLeuAlaGlyGlnGlyAlaAla 253035
GCGCTGGTGGGGGGCGTGCTGCTCATCGGCGCGGTGCTCGCGGGGAAC259
AlaLeuValGlyGlyValLeuLeuIleGlyAlaValLeuAlaGlyAsn 404550
TCGCTCGTGTGCGTGAGCGTGGCCACCGAGCGCGCCCTGCAGACGCCC307
SerLeuValCysValSerValAlaThrGluArgAlaLeuGlnThrPro 556065
ACCAACTCCTTCATCGTGAGCCTGGCGGCCGCCGACCTCCTCCTCGCT355
ThrAsnSerPheIleValSerLeuAlaAlaAlaAspLeuLeuLeuAla 707580
CTCCTGGTGCTGCCGCTCTTCGTCTACTCCGAGGTCCAGGGTGGCGCG403
LeuLeuValLeuProLeuPheValTyrSerGluValGlnGlyGlyAla 859095100
TGGCTGCTGAGCCCCCGCCTGTGCGACGCCCTCATGGCCATGGACGTC451
TrpLeuLeuSerProArgLeuCysAspAlaLeuMetAlaMetAspVal 105110115
ATGCTGTGCACCGCCTCCATCTTCAACCTGTGCGCCATCAGCGTGGAC499
MetLeuCysThrAlaSerIlePheAsnLeuCysAlaIleSerValAsp 120125130
AGGTTCGTGGCCGTGGCCGTGCCGCTGCGCTACAACCGGCAGGGTGGG547
ArgPheValAlaValAlaValProLeuArgTyrAsnArgGlnGlyGly 135140145
AGCCGCCGGCAGCTGCTGCTCATCGGCGCCACGTGGCTGCTGTCCGCG595
SerArgArgGlnLeuLeuLeuIleGlyAlaThrTrpLeuLeuSerAla 150155160
GCGGTGGCGGCGCCCGTACTGTGCGGCCTCAACGACGTGCGCGGCCGC643
AlaValAlaAlaProValLeuCysGlyLeuAsnAspValArgGlyArg 165170175180
GACCCCGCCGTGTGCCGCCTGGAGGACCGCGACTACGTGGTCTACTCG691
AspProAlaValCysArgLeuGluAspArgAspTyrValValTyrSer 185190195
TCCGTGTGCTCCTTCTTCCTACCCTGCCCGCTCATGCTGCTGCTGTAC739
SerValCysSerPhePheLeuProCysProLeuMetLeuLeuLeuTyr 200205210
TGGGCCACGTTCCGCGGCCTGCAGCGCTGGGAGGTGGCACGTCGCGCC787
TrpAlaThrPheArgGlyLeuGlnArgTrpGluValAlaArgArgAla 215220225
AAGCTGCACGGCCGCGCGCCCCGCCGACCCAGCGGCCCTGGCCCGCCT835
LysLeuHisGlyArgAlaProArgArgProSerGlyProGlyProPro 230235240
TCCCCCACGCCACCCGCGCCCCGCCTCCCCCAGGACCCCTGCGGCCCC883
SerProThrProProAlaProArgLeuProGlnAspProCysGlyPro 245250255260
GACTGTGCGCCCCCCGCGCCCGGCCTCCCCCCGGACCCCTGCGGCTCC931
AspCysAlaProProAlaProGlyLeuProProAspProCysGlySer 265270275
AACTGTGCTCCCCCCGACGCCGTCAGAGCCGCCGCGCTCCCACCCCAG979
AsnCysAlaProProAspAlaValArgAlaAlaAlaLeuProProGln 280285290
ACTCCACCGCAGACCCGCAGGAGGCGGCGTGCCAAGATCACCGGCCGG1027
ThrProProGlnThrArgArgArgArgArgAlaLysIleThrGlyArg 295300305
GAGCGCAAGGCCATGAGGGTCCTGCCGGTGGTGGTCGGGGCCTTCCTG1075
GluArgLysAlaMetArgValLeuProValValValGlyAlaPheLeu 310315320
CTGTGCTGGACGCCCTTCTTCGTGGTGCACATCACGCAGGCGCTGTGT1123
LeuCysTrpThrProPhePheValValHisIleThrGlnAlaLeuCys 325330335340
CCTGCCTGCTCCGTGCCCCCGCGGCTGGTCAGCGCCGTCACCTGGCTG1171
ProAlaCysSerValProProArgLeuValSerAlaValThrTrpLeu 345350355
GGCTACGTCAACAGCGCCCTCACCCCCGTCATCTACACTGTCTTCAAC1219
GlyTyrValAsnSerAlaLeuThrProValIleTyrThrValPheAsn 360365370
GCCGAGTTCCGCAACGTCTTCCGCAAGGCCCTGCGTGCCTGCTGCTGAGCCGG1274
AlaGluPheArgAsnValPheArgLysAlaLeuArgAlaCysCys 375380385
ACCCCCGGACGCCCCCCGGCCTGATGGCCAGGCCTCAGGGACCAAGGAGATGGGGAGGGC1334
GCTTTTGTACGTTAATTAAACAAATTCCTTCCCAAA1370 (2) INFORMATION FOR SEQ ID
NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 387 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
MetGlyAsnArgSerThrAlaAspAlaAspGlyLeuLeuAlaGlyArg 151015
GlyArgAlaAlaGlyAlaSerAlaGlyAlaSerAlaGlyLeuAlaGly 202530
GlnGlyAlaAlaAlaLeuValGlyGlyValLeuLeuIleGlyAlaVal 354045
LeuAlaGlyAsnSerLeuValCysValSerValAlaThrGluArgAla 505560
LeuGlnThrProThrAsnSerPheIleValSerLeuAlaAlaAlaAsp 65707580
LeuLeuLeuAlaLeuLeuValLeuProLeuPheValTyrSerGluVal 859095
GlnGlyGlyAlaTrpLeuLeuSerProArgLeuCysAspAlaLeuMet 100105110
AlaMetAspValMetLeuCysThrAlaSerIlePheAsnLeuCysAla 115120125
IleSerValAspArgPheValAlaValAlaValProLeuArgTyrAsn 130135140
ArgGlnGlyGlySerArgArgGlnLeuLeuLeuIleGlyAlaThrTrp 145150155160
LeuLeuSerAlaAlaValAlaAlaProValLeuCysGlyLeuAsnAsp 165170175
ValArgGlyArgAspProAlaValCysArgLeuGluAspArgAspTyr 180185190
ValValTyrSerSerValCysSerPhePheLeuProCysProLeuMet 195200205
LeuLeuLeuTyrTrpAlaThrPheArgGlyLeuGlnArgTrpGluVal 210215220
AlaArgArgAlaLysLeuHisGlyArgAlaProArgArgProSerGly 225230235240
ProGlyProProSerProThrProProAlaProArgLeuProGlnAsp 245250255
ProCysGlyProAspCysAlaProProAlaProGlyLeuProProAsp 260265270
ProCysGlySerAsnCysAlaProProAspAlaValArgAlaAlaAla
275280285 LeuProProGlnThrProProGlnThrArgArgArgArgArgAlaLys
290295300 IleThrGlyArgGluArgLysAlaMetArgValLeuProValValVal
305310315320 GlyAlaPheLeuLeuCysTrpThrProPhePheValValHisIleThr
325330335 GlnAlaLeuCysProAlaCysSerValProProArgLeuValSerAla
340345350 ValThrTrpLeuGlyTyrValAsnSerAlaLeuThrProValIleTyr
355360365 ThrValPheAsnAlaGluPheArgAsnValPheArgLysAlaLeuArg
370375380 AlaCysCys 385 (2) INFORMATION FOR SEQ ID NO:19: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 1466 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: 5'UTR (B) LOCATION:
1..103 (ix) FEATURE: (A) NAME/KEY: 3'UTR (B) LOCATION: 1364..1466
(ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 104..1363 (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:19:
CGGGGGCGGGACCAGGGTCCGGCCGGGGCGTGCCCCCGGGGAGGGACTCCCCGGCTTGCC60
CCCCGGCGTTGTCCGCGGTGCTCAGCGCCCGCCCGGGCGCGCCATGGGGAACCGC115
MetGlyAsnArg 1 AGCACCGCGGACGCGGACGGGCTGCTGGCTGGGCGCGGGCGGGCCGCG163
SerThrAlaAspAlaAspGlyLeuLeuAlaGlyArgGlyArgAlaAla 5101520
GGGGCATCTGCGGGGGCATCTGCGGGGCTGGCTGGGCAGGGCGCGGCG211
GlyAlaSerAlaGlyAlaSerAlaGlyLeuAlaGlyGlnGlyAlaAla 253035
GCGCTGGTGGGGGGCGTGCTGCTCATCGGCGCGGTGCTCGCGGGGAAC259
AlaLeuValGlyGlyValLeuLeuIleGlyAlaValLeuAlaGlyAsn 404550
TCGCTCGTGTGCGTGAGCGTGGCCACCGAGCGCGCCCTGCAGACGCCC307
SerLeuValCysValSerValAlaThrGluArgAlaLeuGlnThrPro 556065
ACCAACTCCTTCATCGTGAGCCTGGCGGCCGCCGACCTCCTCCTCGCT355
ThrAsnSerPheIleValSerLeuAlaAlaAlaAspLeuLeuLeuAla 707580
CTCCTGGTGCTGCCGCTCTTCGTCTACTCCGAGGTCCAGGGTGGCGCG403
LeuLeuValLeuProLeuPheValTyrSerGluValGlnGlyGlyAla 859095100
TGGCTGCTGAGCCCCCGCCTGTGCGACGCCCTCATGGCCATGGACGTC451
TrpLeuLeuSerProArgLeuCysAspAlaLeuMetAlaMetAspVal 105110115
ATGCTGTGCACCGCCTCCATCTTCAACCTGTGCGCCATCAGCGTGGAC499
MetLeuCysThrAlaSerIlePheAsnLeuCysAlaIleSerValAsp 120125130
AGGTTCGTGGCCGTGGCCGTGCCGCTGCGCTACAACCGGCAGGGTGGG547
ArgPheValAlaValAlaValProLeuArgTyrAsnArgGlnGlyGly 135140145
AGCCGCCGGCAGCTGCTGCTCATCGGCGCCACGTGGCTGCTGTCCGCG595
SerArgArgGlnLeuLeuLeuIleGlyAlaThrTrpLeuLeuSerAla 150155160
GCGGTGGCGGCGCCCGTACTGTGCGGCCTCAACGACGTGCGCGGCCGC643
AlaValAlaAlaProValLeuCysGlyLeuAsnAspValArgGlyArg 165170175180
GACCCCGCCGTGTGCCGCCTGGAGGACCGCGACTACGTGGTCTACTCG691
AspProAlaValCysArgLeuGluAspArgAspTyrValValTyrSer 185190195
TCCGTGTGCTCCTTCTTCCTACCCTGCCCGCTCATGCTGCTGCTGTAC739
SerValCysSerPhePheLeuProCysProLeuMetLeuLeuLeuTyr 200205210
TGGGCCACGTTCCGCGGCCTGCAGCGCTGGGAGGTGGCACGTCGCGCC787
TrpAlaThrPheArgGlyLeuGlnArgTrpGluValAlaArgArgAla 215220225
AAGCTGCACGGCCGCGCGCCCCGCCGACCCAGCGGCCCTGGCCCGCCT835
LysLeuHisGlyArgAlaProArgArgProSerGlyProGlyProPro 230235240
TCCCCCACGCCACCCGCGCCCCGCCTCCCCCAGGACCCCTGCGGCCCC883
SerProThrProProAlaProArgLeuProGlnAspProCysGlyPro 245250255260
GACTGTGCGCCCCCCGCGCCCGGCCTTCCCCGGGGTCCCTGCGGCCCC931
AspCysAlaProProAlaProGlyLeuProArgGlyProCysGlyPro 265270275
GACTGTGCGCCCGCCGCGCCCAGCCTCCCCCAGGACCCCTGCGGCCCC979
AspCysAlaProAlaAlaProSerLeuProGlnAspProCysGlyPro 280285290
GACTGTGCGCCCCCCGCGCCCGGCCTCCCCCCGGACCCCTGCGGCTCC1027
AspCysAlaProProAlaProGlyLeuProProAspProCysGlySer 295300305
AACTGTGCTCCCCCCGACGCCGTCAGAGCCGCCGCGCTCCCACCCCAG1075
AsnCysAlaProProAspAlaValArgAlaAlaAlaLeuProProGln 310315320
ACTCCACCGCAGACCCGCAGGAGGCGGCGTGCCAAGATCACCGGCCGG1123
ThrProProGlnThrArgArgArgArgArgAlaLysIleThrGlyArg 325330335340
GAGCGCAAGGCCATGAGGGTCCTGCCGGTGGTGGTCGGGGCCTTCCTG1171
GluArgLysAlaMetArgValLeuProValValValGlyAlaPheLeu 345350355
CTGTGCTGGACGCCCTTCTTCGTGGTGCACATCACGCAGGCGCTGTGT1219
LeuCysTrpThrProPhePheValValHisIleThrGlnAlaLeuCys 360365370
CCTGCCTGCTCCGTGCCCCCGCGGCTGGTCAGCGCCGTCACCTGGCTG1267
ProAlaCysSerValProProArgLeuValSerAlaValThrTrpLeu 375380385
GGCTACGTCAACAGCGCCCTCACCCCCGTCATCTACACTGTCTTCAAC1315
GlyTyrValAsnSerAlaLeuThrProValIleTyrThrValPheAsn 390395400
GCCGAGTTCCGCAACGTCTTCCGCAAGGCCCTGCGTGCCTGCTGCTGAGCCGG1370
AlaGluPheArgAsnValPheArgLysAlaLeuArgAlaCysCys 405410415420
ACCCCCGGACGCCCCCCGGCCTGATGGCCAGGCCTCAGGGACCAAGGAGATGGGGAGGGC1430
GCTTTTGTACGTTAATTAAACAAATTCCTTCCCAAA1466 (2) INFORMATION FOR SEQ ID
NO:20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 419 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
MetGlyAsnArgSerThrAlaAspAlaAspGlyLeuLeuAlaGlyArg 151015
GlyArgAlaAlaGlyAlaSerAlaGlyAlaSerAlaGlyLeuAlaGly 202530
GlnGlyAlaAlaAlaLeuValGlyGlyValLeuLeuIleGlyAlaVal 354045
LeuAlaGlyAsnSerLeuValCysValSerValAlaThrGluArgAla 505560
LeuGlnThrProThrAsnSerPheIleValSerLeuAlaAlaAlaAsp 65707580
LeuLeuLeuAlaLeuLeuValLeuProLeuPheValTyrSerGluVal 859095
GlnGlyGlyAlaTrpLeuLeuSerProArgLeuCysAspAlaLeuMet 100105110
AlaMetAspValMetLeuCysThrAlaSerIlePheAsnLeuCysAla 115120125
IleSerValAspArgPheValAlaValAlaValProLeuArgTyrAsn 130135140
ArgGlnGlyGlySerArgArgGlnLeuLeuLeuIleGlyAlaThrTrp 145150155160
LeuLeuSerAlaAlaValAlaAlaProValLeuCysGlyLeuAsnAsp 165170175
ValArgGlyArgAspProAlaValCysArgLeuGluAspArgAspTyr 180185190
ValValTyrSerSerValCysSerPhePheLeuProCysProLeuMet 195200205
LeuLeuLeuTyrTrpAlaThrPheArgGlyLeuGlnArgTrpGluVal 210215220
AlaArgArgAlaLysLeuHisGlyArgAlaProArgArgProSerGly 225230235240
ProGlyProProSerProThrProProAlaProArgLeuProGlnAsp 245250255
ProCysGlyProAspCysAlaProProAlaProGlyLeuProArgGly 260265270
ProCysGlyProAspCysAlaProAlaAlaProSerLeuProGlnAsp 275280285
ProCysGlyProAspCysAlaProProAlaProGlyLeuProProAsp 290295300
ProCysGlySerAsnCysAlaProProAspAlaValArgAlaAlaAla 305310315320
LeuProProGlnThrProProGlnThrArgArgArgArgArgAlaLys 325330335
IleThrGlyArgGluArgLysAlaMetArgValLeuProValValVal 340345350
GlyAlaPheLeuLeuCysTrpThrProPhePheValValHisIleThr 355360365
GlnAlaLeuCysProAlaCysSerValProProArgLeuValSerAla 370375380
ValThrTrpLeuGlyTyrValAsnSerAlaLeuThrProValIleTyr 385390395400
ThrValPheAsnAlaGluPheArgAsnValPheArgLysAlaLeuArg 405410415
AlaCysCys (2) INFORMATION FOR SEQ ID NO:21: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 1610 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (ix) FEATURE: (A) NAME/KEY: 5'UTR (B) LOCATION: 1..103 (ix)
FEATURE: (A) NAME/KEY: 3'UTR (B) LOCATION: 1508..1610 (ix) FEATURE:
(A) NAME/KEY: CDS (B) LOCATION: 104..1507 (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:21:
CGGGGGCGGGACCAGGGTCCGGCCGGGGCGTGCCCCCGGGGAGGGACTCCCCGGCTTGCC60
CCCCGGCGTTGTCCGCGGTGCTCAGCGCCCGCCCGGGCGCGCCATGGGGAACCGC115
MetGlyAsnArg 1 AGCACCGCGGACGCGGACGGGCTGCTGGCTGGGCGCGGGCGGGCCGCG163
SerThrAlaAspAlaAspGlyLeuLeuAlaGlyArgGlyArgAlaAla 5101520
GGGGCATCTGCGGGGGCATCTGCGGGGCTGGCTGGGCAGGGCGCGGCG211
GlyAlaSerAlaGlyAlaSerAlaGlyLeuAlaGlyGlnGlyAlaAla 253035
GCGCTGGTGGGGGGCGTGCTGCTCATCGGCGCGGTGCTCGCGGGGAAC259
AlaLeuValGlyGlyValLeuLeuIleGlyAlaValLeuAlaGlyAsn 404550
TCGCTCGTGTGCGTGAGCGTGGCCACCGAGCGCGCCCTGCAGACGCCC307
SerLeuValCysValSerValAlaThrGluArgAlaLeuGlnThrPro 556065
ACCAACTCCTTCATCGTGAGCCTGGCGGCCGCCGACCTCCTCCTCGCT355
ThrAsnSerPheIleValSerLeuAlaAlaAlaAspLeuLeuLeuAla 707580
CTCCTGGTGCTGCCGCTCTTCGTCTACTCCGAGGTCCAGGGTGGCGCG403
LeuLeuValLeuProLeuPheValTyrSerGluValGlnGlyGlyAla 859095100
TGGCTGCTGAGCCCCCGCCTGTGCGACGCCCTCATGGCCATGGACGTC451
TrpLeuLeuSerProArgLeuCysAspAlaLeuMetAlaMetAspVal 105110115
ATGCTGTGCACCGCCTCCATCTTCAACCTGTGCGCCATCAGCGTGGAC499
MetLeuCysThrAlaSerIlePheAsnLeuCysAlaIleSerValAsp 120125130
AGGTTCGTGGCCGTGGCCGTGCCGCTGCGCTACAACCGGCAGGGTGGG547
ArgPheValAlaValAlaValProLeuArgTyrAsnArgGlnGlyGly 135140145
AGCCGCCGGCAGCTGCTGCTCATCGGCGCCACGTGGCTGCTGTCCGCG595
SerArgArgGlnLeuLeuLeuIleGlyAlaThrTrpLeuLeuSerAla 150155160
GCGGTGGCGGCGCCCGTACTGTGCGGCCTCAACGACGTGCGCGGCCGC643
AlaValAlaAlaProValLeuCysGlyLeuAsnAspValArgGlyArg 165170175180
GACCCCGCCGTGTGCCGCCTGGAGGACCGCGACTACGTGGTCTACTCG691
AspProAlaValCysArgLeuGluAspArgAspTyrValValTyrSer 185190195
TCCGTGTGCTCCTTCTTCCTACCCTGCCCGCTCATGCTGCTGCTGTAC739
SerValCysSerPhePheLeuProCysProLeuMetLeuLeuLeuTyr 200205210
TGGGCCACGTTCCGCGGCCTGCAGCGCTGGGAGGTGGCACGTCGCGCC787
TrpAlaThrPheArgGlyLeuGlnArgTrpGluValAlaArgArgAla 215220225
AAGCTGCACGGCCGCGCGCCCCGCCGACCCAGCGGCCCTGGCCCGCCT835
LysLeuHisGlyArgAlaProArgArgProSerGlyProGlyProPro 230235240
TCCCCCACGCCACCCGCGCCCCGCCTCCCCCAGGACCCCTGCGGCCCC883
SerProThrProProAlaProArgLeuProGlnAspProCysGlyPro 245250255260
GACTGTGCGCCCCCCGCGCCCGGCCTTCCCCGGGGTCCCTGCGGCCCC931
AspCysAlaProProAlaProGlyLeuProArgGlyProCysGlyPro 265270275
GACTGTGCGCCCGCCGCGCCCGGCCTCCCCCCGGACCCCTGCGGCCCC979
AspCysAlaProAlaAlaProGlyLeuProProAspProCysGlyPro 280285290
GACTGTGCGCCCCCCGCGCCCGGCCTCCCCCAGGACCCCTGCGGCCCC1027
AspCysAlaProProAlaProGlyLeuProGlnAspProCysGlyPro 295300305
GACTGTGCGCCCCCCGCGCCCGGCCTTCCCCGGGGTCCCTGCGGCCCC1075
AspCysAlaProProAlaProGlyLeuProArgGlyProCysGlyPro 310315320
GACTGTGCGCCCCCCGCGCCCGGCCTCCCCCAGGACCCCTGCGGCCCC1123
AspCysAlaProProAlaProGlyLeuProGlnAspProCysGlyPro 325330335340
GACTGTGCGCCCCCCGCGCCCGGCCTCCCCCCGGACCCCTGCGGCTCC1171
AspCysAlaProProAlaProGlyLeuProProAspProCysGlySer 345350355
AACTGTGCTCCCCCCGACGCCGTCAGAGCCGCCGCGCTCCCACCCCAG1219
AsnCysAlaProProAspAlaValArgAlaAlaAlaLeuProProGln 360365370
ACTCCACCGCAGACCCGCAGGAGGCGGCGTGCCAAGATCACCGGCCGG1267
ThrProProGlnThrArgArgArgArgArgAlaLysIleThrGlyArg 375380385
GAGCGCAAGGCCATGAGGGTCCTGCCGGTGGTGGTCGGGGCCTTCCTG1315
GluArgLysAlaMetArgValLeuProValValValGlyAlaPheLeu 390395400
CTGTGCTGGACGCCCTTCTTCGTGGTGCACATCACGCAGGCGCTGTGT1363
LeuCysTrpThrProPhePheValValHisIleThrGlnAlaLeuCys 405410415420
CCTGCCTGCTCCGTGCCCCCGCGGCTGGTCAGCGCCGTCACCTGGCTG1411
ProAlaCysSerValProProArgLeuValSerAlaValThrTrpLeu 425430435
GGCTACGTCAACAGCGCCCTCACCCCCGTCATCTACACTGTCTTCAAC1459
GlyTyrValAsnSerAlaLeuThrProValIleTyrThrValPheAsn 440445450
GCCGAGTTCCGCAACGTCTTCCGCAAGGCCCTGCGTGCCTGCTGCTGAGCCGG1514
AlaGluPheArgAsnValPheArgLysAlaLeuArgAlaCysCys 455460465
ACCCCCGGACGCCCCCCGGCCTGATGGCCAGGCCTCAGGGACCAAGGAGATGGGGAGGGC1574
GCTTTTGTACGTTAATTAAACAAATTCCTTCCCAAA1610 (2) INFORMATION FOR SEQ ID
NO:22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 467 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
MetGlyAsnArgSerThrAlaAspAlaAspGlyLeuLeuAlaGlyArg 151015
GlyArgAlaAlaGlyAlaSerAlaGlyAlaSerAlaGlyLeuAlaGly 202530
GlnGlyAlaAlaAlaLeuValGlyGlyValLeuLeuIleGlyAlaVal 354045
LeuAlaGlyAsnSerLeuValCysValSerValAlaThrGluArgAla 505560
LeuGlnThrProThrAsnSerPheIleValSerLeuAlaAlaAlaAsp 65707580
LeuLeuLeuAlaLeuLeuValLeuProLeuPheValTyrSerGluVal 859095
GlnGlyGlyAlaTrpLeuLeuSerProArgLeuCysAspAlaLeuMet 100105110
AlaMetAspValMetLeuCysThrAlaSerIlePheAsnLeuCysAla 115120125
IleSerValAspArgPheValAlaValAlaValProLeuArgTyrAsn 130135140
ArgGlnGlyGlySerArgArgGlnLeuLeuLeuIleGlyAlaThrTrp 145150155160
LeuLeuSerAlaAlaValAlaAlaProValLeuCysGlyLeuAsnAsp 165170175
ValArgGlyArgAspProAlaValCysArgLeuGluAspArgAspTyr 180185190
ValValTyrSerSerValCysSerPhePheLeuProCysProLeuMet 195200205
LeuLeuLeuTyrTrpAlaThrPheArgGlyLeuGlnArgTrpGluVal 210215220
AlaArgArgAlaLysLeuHisGlyArgAlaProArgArgProSerGly 225230235240
ProGlyProProSerProThrProProAlaProArgLeuProGlnAsp 245250255
ProCysGlyProAspCysAlaProProAlaProGlyLeuProArgGly 260265270
ProCysGlyProAspCysAlaProAlaAlaProGlyLeuProProAsp 275280285
ProCysGlyProAspCysAlaProProAlaProGlyLeuProGlnAsp 290295300
ProCysGlyProAspCysAlaProProAlaProGlyLeuProArgGly 305310315320
ProCysGlyProAspCysAlaProProAlaProGlyLeuProGlnAsp 325330335
ProCysGlyProAspCysAlaProProAlaProGlyLeuProProAsp 340345350
ProCysGlySerAsnCysAlaProProAspAlaValArgAlaAlaAla 355360365
LeuProProGlnThrProProGlnThrArgArgArgArgArgAlaLys 370375380
IleThrGlyArgGluArgLysAlaMetArgValLeuProValValVal 385390395400
GlyAlaPheLeuLeuCysTrpThrProPhePheValValHisIleThr 405410415
GlnAlaLeuCysProAlaCysSerValProProArgLeuValSerAla 420425430
ValThrTrpLeuGlyTyrValAsnSerAlaLeuThrProValIleTyr 435440445
ThrValPheAsnAlaGluPheArgAsnValPheArgLysAlaLeuArg 450455460
AlaCysCys 465
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